Novel t cell receptors and immune therapy using the same

ABSTRACT

The present invention pertains to antigen recognizing constructs against tumor associated antigens (TAA), in particular the TAA Serine protease inhibitor Kazal-type 2 (SPINK2). The invention in particular provides novel T cell receptor (TCR) based molecules which are selective and specific for the tumor expressed antigen of the invention. The TCR of the invention, and SPINK2 binding fragments derived therefrom, are of use for the diagnosis, treatment and prevention of SPINK2 expressing cancerous diseases. Further provided are nucleic acids encoding the antigen recognizing constructs of the invention, vectors comprising these nucleic acids, recombinant cells expressing the antigen recognizing constructs and pharmaceutical compositions comprising the compounds of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/023,731, filed 29 Jun. 2018, which claims priority to 62/527,844,filed 30 Jun. 2017 and German Patent Application No. 10 2017 114 737.3,filed 30 Jun. 2017. The disclosures of the priority applications areincorporated in their entirety herein by reference.

This application is also related to PCT/EP2018/067380, filed 28 Jun.2018, the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “3000058-010003_Sequence_Listing_ST25.txt” createdon 26 Dec. 2019, and 127,719 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention pertains to antigen recognizing constructs againsttumor associated antigens (TAA), in particular the TAA Serine proteaseinhibitor Kazal-type 2 (SPINK2). The invention in particular providesnovel T cell receptor (TCR) based molecules which are selective andspecific for the tumor expressed antigen of the invention. The TCR ofthe invention, and SPINK2 binding fragments derived therefrom, are ofuse for the diagnosis, treatment and prevention of SPINK2 expressingcancerous diseases. Further provided are nucleic acids encoding theantigen recognizing constructs of the invention, vectors comprisingthese nucleic acids, recombinant cells expressing the antigenrecognizing constructs and pharmaceutical compositions comprising thecompounds of the invention.

DESCRIPTION OF RELATED ART

Serine protease inhibitor Kazal-type 2 (SPINK2), is also known asacrosin-trypsin inhibitor, and is a protein that in humans is encoded bythe SPINK2 gene. The encoded protein acts as a trypsin and acrosininhibitor in the genital tract and is localized in the spermatozoa. Theprotein has been associated with the progression of lymphomas.Alternative splicing results in multiple transcript variants.

T-cell based immunotherapy targets represent peptide epitopes derivedfrom tumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). These tumorassociated antigens (TAAs) can be peptides derived from all proteinclasses, such as enzymes, receptors, transcription factors, etc. whichare expressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defective ribosomal products (DRiPs) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

There are two classes of MHC-molecules, MHC class I and MHC class II.Complexes of peptide and MHC class I are recognized by CD8-positiveT-cells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T-cells bearing the appropriate TCR. Since bothtypes of response, CD8 and CD4 dependent, contribute jointly andsynergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens and corresponding T cellreceptors is important in the development of cancer immunotherapies suchas vaccines and cell therapies.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T-cells bearingspecific T-cell receptors (TCR). Therefore, TAAs are a starting pointfor the development of a T-cell based therapy including but not limitedto tumor vaccines and cell therapies.

Approximately 90 percent of peripheral blood T cells express a TCRconsisting of an α polypeptide and a β polypeptide. A small percentageof T cells (about 5% of total T cells) have been shown to express a TCRconsisting of a γ polypeptide and a δ polypeptide. γδ T cells are foundat their highest abundance in the gut mucosa, within a population oflymphocytes known as intraepithelial lymphocytes (IELs). The antigenicmolecules that activate γδ T cells are still widely unknown. However, γδT cells are not MHC restricted and seem to be able to recognize wholeproteins rather than requiring peptides to be presented by MHC moleculeson antigen presenting cells, although some recognize MHC class IBmolecules. Human Vγ9/Vδ2 T cells, which constitute the major γδ T cellpopulation in peripheral blood, are unique in that they specifically andrapidly respond to a small non-peptidic microbial metabolite, HMB-PP, anisopentenyl pyrophosphate precursor. Estimates of the percentages of Tcells that may be found in peripheral blood from healthy donors are asfollows: CD3+=70.78%±4.71; CD3+CD4=38.97%±5.66; CD3+CD8=28.955%±7.43;CD3+CD56+=5.22%±1.74; CD3−CD56+=10.305%±4.7; CD3+CD45RA=45.00%±7.19;CD3+CD45RO+=27.21%±7.34.

The chains of the T cell antigen receptor of a T cell clone are eachcomposed of a unique combination of domains designated variable (V),[diversity (D),] joining (J), and constant (C). In each T cell clone,the combination of V, D and J domains of both the alpha and the betachains or of both the delta and gamma chains participates in antigenrecognition in a manner which is uniquely characteristic of that T cellclone and defines a unique binding site, also known as the idiotype ofthe T cell clone. In contrast, the C domain does not participate inantigen binding.

A TCR is a heterodimeric cell surface protein of the immunoglobulinsuper-family, which is associated with invariant proteins of the CD3complex involved in mediating signal transduction. TCRs exist in αβ andγδ forms, which are structurally similar but have quite distinctanatomical locations and probably functions. The extracellular portionof native heterodimeric αβTCR and γδTCR each contain two polypeptides,each of which has a membrane-proximal constant domain, and amembrane-distal variable domain. Each of the constant and variabledomains includes an intra-chain disulfide bond. The variable domainscontain the highly polymorphic loops analogous to the complementaritydetermining regions (CDRs) of antibodies. The use of TCR gene therapyovercomes a number of current hurdles. It allows equipping patients' ownT cells with desired specificities and generation of sufficient numbersof T cells in a short period of time, avoiding their exhaustion. The TCRwill be transduced into central memory T cells or T cells with stem cellcharacteristics, which may ensure better persistence and function upontransfer. TCR-engineered T cells will be infused into cancer patientsrendered lymphopenic by chemotherapy or irradiation, allowing efficientengraftment but inhibiting immune suppression.

While advances have been made in the development of molecular-targetingdrugs for cancer therapy, there remains a need in the art to develop newanti-cancer agents that specifically target molecules highly specific tocancer cells. The present description addresses that need by providingnovel SPINK2-001 TCRs, respective recombinant TCR constructs, nucleicacids, vectors and host cells that specifically bind TAA epitope(s) asdisclosed; and methods of using such molecules in the treatment ofcancer. The term TAA in context of the invention relates in particularto the protein SPINK2 and even more preferably to the epitope SPINK2-001as also disclosed herein elsewhere.

SUMMARY Antigen Recognizing Constructs

The object of the invention is solved in a first aspect by an antigenrecognizing construct comprising at least one complementary determiningregion (CDR) 3 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,or preferably 100% sequence identity to an amino acid sequence selectedfrom SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81,87, 93, 99, 105, 111, and 117.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1-33 depict embodiments of the disclosure as described herein.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

In some embodiments the antigen recognizing construct of the inventionspecifically binds to a TAA-peptide-HLA molecule complex, wherein theTAA peptide comprises, or alternatively consists of, a variant of theTAA which is at least 66%, preferably at least 77%, and more preferablyat least 88% homologous (preferably at least 77% or at least 88%identical) to the amino acid sequence of the TAA of the invention,wherein said variant binds to an HLA class I or class II molecule and/orinduces T-cells cross-reacting with said peptide, or a pharmaceuticallyacceptable salt thereof, wherein said peptide is not the underlyingfull-length polypeptide.

As used herein, the terms “identical” or percent “identity”, when usedanywhere herein in the context of two or more nucleic acid orprotein/polypeptide sequences, refer to two or more sequences orsubsequences that are the same or have (or have at least) a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., at, or at least, about 60% identity, preferably at, or at least,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94%, identity, and morepreferably at, or at least, about 95%, 96%, 97%, 98%, 99%, or higheridentity over a specified region—preferably over their full lengthsequences—, when compared and aligned for maximum correspondence overthe comparison window or designated region) as measured using a sequencecomparison algorithms, or by manual alignment and visual inspection(see, e.g., NCBI web site). In a particular embodiment, for example whencomparing the protein or nucleic acid sequence of an antigen recognizingconstruct of the invention to another protein/gene, the percentageidentity can be determined by the Blast searches supported at the NCBIweb site; in particular for amino acid identity, those using BLASTP withthe following parameters: Expected threshold 10; Word size: 6; Matrix:BLOSUM62; Gap Costs: Existence: 11, Extension: 1; Neighboring wordsthreshold: 11; Compositional adjustments: Conditional compositionalscore matrix adjustment.

In the context of the present invention it shall be understood that anyembodiments referred to as “comprising” certain features of theinvention, shall be understood to include in some more preferredembodiments the more restricted description of “consisting of” or“consisting essentially of” the very same features of the presentinvention.

In another additional or alternative embodiment, the antigen recognizingconstruct may further comprise a CDR1 and/or a CDR2 domain sequence.Within the variable domain, CDR1 and CDR2 are found in the variable (V)region of a polypeptide chain, and CDR3 includes some of V, all ofdiversity (D) and joining (J) regions. CDR3 is the most variable and isthe main CDR responsible for specifically and selectively recognizing anantigen. CDR1 and CDR2 sequences may be selected from a CDR sequence ofa human variable chain allele.

Native alpha-beta heterodimeric TCRs have an alpha chain and a betachain. Each chain comprises variable, joining and constant regions, andthe beta chain also usually contains a short diversity region betweenthe variable and joining regions, but this diversity region is oftenconsidered as part of the joining region. Each variable region comprisesthree CDRs (Complementarity Determining Regions) embedded in a frameworksequence, one being the hypervariable region named CDR3. There areseveral types of alpha chain variable (Va) regions and several types ofbeta chain variable (Vβ) regions distinguished by their framework, CDR1and CDR2 sequences, and by a partly defined CDR3 sequence. The Va typesare referred to in IMGT nomenclature by a unique TRAV number, VP typesare referred to by a unique TRBV number. For more information onimmunoglobulin antibody and TCR genes see the internationalImMunoGeneTics information System®, Lefranc M-P et al (Nucleic AcidsRes. 2015 January; 43(Database issue):D413-22; andhttp://www.imgt.org/).

Therefore, in one additional or alternative embodiment the antigenrecognizing construct of the invention comprises CDR1, CDR2 and CDR3sequences in a combination as provided in table 1 herein below, whichdisplay the respective variable chain allele together with the CDR3sequence. Therefore, preferred are antigen recognizing constructs of theinvention which comprise at least one, preferably, all three CDRsequences CDR1, CDR2 and CDR3. Preferably, an antigen recognizingconstruct of the invention comprises the respective CDR1 to CDR3 of oneindividual herein disclosed TCR variable region of the invention (seetable 1 herein below and the example section).

The term “specificity” or “antigen specificity” or “specific for” agiven antigen, as used herein means that the antigen recognizingconstruct can specifically bind to said antigen, preferably a TAAantigen, more preferably with high avidity, when said antigen ispresented by HLA, preferably by HLA-A*02. For example, a TCR, as antigenrecognizing construct, may be considered to have “antigenic specificity”for the TAA, if T cells expressing the TCR and contacted with a TAApresenting HLA secrete at least about 200 pg/ml or more (e.g., 250 pg/mlor more, 300 pg/ml or more, 400 pg/ml or more, 500 pg/ml or more, 600pg/ml or more, 700 pg/ml or more, 1000 pg ml or more, 2,000 pg/ml ormore, 2,500 pg/ml or more, 5,000 pg/ml or more) of interferon γ (IFN-γ)upon co-culture with target cells pulsed with a low concentration of aTAA antigen, such as the TAA epitopes and antigens provided herein below(e.g., about 10⁻¹¹ mol/l, 10⁻¹⁰ mol/1, 10⁻⁹ mol/1, 10⁻⁸ mol/1, 10⁻⁷mol/1, 10⁻⁶ mol/1, 10⁻⁵ mol/1). Alternatively, or additionally, a TCRmay be considered to have “antigenic specificity” for the TAA, if Tcells expressing the TCR secrete at least twice as much IFN-γ as theuntransduced background level of IFN-γ upon co-culture with target cellspulsed with a low concentration of the TAA antigens. Such a“specificity” as described above can—for example—be analyzed with anELISA.

In one alternative or additional embodiment of the invention, theantigen recognizing construct selectively binds to a TAA derivedantigenic peptide; preferably wherein the TAA antigenic peptide is aprotein epitope or peptide having an amino acid sequence shown in table2 below, in particular the antigenic peptide is the SPINK2-001 peptide(SEQ ID NO: 133), or a variant thereof, wherein the variant is an aminoacid deletion, addition, insertion or substitution of not more thanthree, preferably two and most preferably not more than one amino acidposition. Preferred variants of SPINK2-001 are shown in table 2 below.

The term “selectivity” or “selective recognizing/binding” is understoodto refer to the property of an antigen recognizing construct, such as aTCR or antibody, to selectively recognize or bind to preferably only onespecific epitope and preferably shows no or substantially nocross-reactivity to another epitope. Preferably “selectivity” or“selective recognizing/binding” means that the antigen recognizingconstruct (e.g. a TCR) selectively recognizes or binds to preferablyonly one specific epitope and preferably shows no or substantially nocross-reactivity to another epitope, wherein said epitope is unique forone protein, such that the antigen recognizing construct shows no orsubstantially no cross-reactivity to another epitope and anotherprotein.

The antigen recognizing construct according to the invention ispreferably selected from an antibody, or derivative or fragment thereof,or a T cell receptor (TCR), or derivative or fragment thereof. Aderivative or fragment of an antibody or TCR of the invention shallpreferably retain the antigen binding/recognizing ability of the parentmolecule, in particular its specificity and/or selectivity as explainedabove. Such binding functionality may be retained by the presence of aCDR3 region as defined herein.

In an embodiment of the invention, the inventive TCRs are able torecognize TAA antigens in a major histocompatibility complex (MHC) classI-dependent manner. “MHC class I-dependent manner,” as used herein,means that the TCR elicits an immune response upon binding to TAAantigens within the context of an MHC class I molecule. The MHC class Imolecule can be any MHC class I molecule known in the art, e.g., HLA-Amolecules. In a preferred embodiment of the invention, the MHC class Imolecule is an HLA-A*02 molecule.

In an aspect, the TCR-elicited immune response or T-cell response mayrefer to the proliferation and activation of effector functions inducedby a peptide in vitro or in vivo. For MHC class I restricted cytotoxic Tcells, for example, effector functions may be lysis of peptide-pulsed,peptide-precursor pulsed or naturally peptide-presenting target cells,secretion of cytokines, preferably Interferon-gamma, TNF-alpha, or IL-2induced by peptide, secretion of effector molecules, for example,granzymes or perforins induced by peptide, or degranulation.

In an aspect, the TCR-elicited immune response or T-cell response mayrefer to the proliferation and activation of effector functions inducedby a peptide in vitro or in vivo. For MHC class I restricted cytotoxic Tcells, for example, effector functions may be lysis of peptide-pulsed,peptide-precursor pulsed or naturally peptide-presenting target cells,secretion of cytokines, preferably Interferon-gamma, TNF-alpha, or IL-2induced by peptide, secretion of effector molecules, for example,granzymes or perforins induced by peptide, or degranulation.

The invention provides both single chain antigen recognizing constructand double chain recognizing constructs.

In an embodiment, the TCR alpha variable domain has at least onemutation relative to a TCR alpha domain shown in Table 1; and/or the TCRbeta variable domain has at least one mutation relative to a TCR alphadomain shown in Table 1. In an embodiment, a TCR comprising at least onemutation in the TCR alpha variable domain and/or TCR beta variabledomain has a binding affinity for, and/or a binding half-life for, a TAApeptide-HLA molecule complex, which is at least double that of a TCRcomprising the unmutated TCR alpha domain and/or unmutated TCR betavariable domain.

The TCR alpha chains of the present description may further comprise aTCR alpha transmembrane domain and/or a TCR alpha intracellular domain.The TCR beta chains of the present description may further comprise aTCR beta transmembrane domain and/or a TCR beta intracellular domain.

The invention in particular provides a TCR as antigen recognizingconstruct, or fragment or derivative thereof. The TCR preferably is ofhuman, which is understood as being generated from a human TCR locus andtherefore comprising human TCR sequences. Furthermore, the TCR of theinvention may be characterized in that it is of human origin andspecifically recognizes a TAA antigen of the invention.

Another embodiment of the invention additionally or alternativelyprovides the antigen recognizing construct described above, whichinduces an immune response, preferably wherein the immune response ischaracterized by an increase in interferon (IFN) γ levels.

TCRs of the invention may be provided as single chain α or β, or γ andδ, molecules, or alternatively as double chain constructs composed ofboth the α and β chain, or γ and δ chain. Furthermore, if in context ofthe present invention any one, two or all CDR regions of a given TCR asdescribed in the present invention, is grafted from one TCR chain typeinto another, it is in some embodiments preferred that such CDRoriginally derived from an α chain could be grafted into a γ or δ chain,and/or such CDR of a β chain could be grafted into a γ or δ chainframework. Thereby it is possible if starting with CDR sequences of anα/β TCR, one can obtain a γ/δ TCR, however, in two ways. One is bygrafting the CDR from α into a γ framework and from β into a δframework. A second, as described above, by grafting the CDR from an αinto a δ framework, and from β into a γ framework. The person of skillis aware of the similarity of the framework regions between the α/β, andγ/δ TCR chains and thus, such embodiments are encompassed by the presentinvention (see also Lefranc M-P et al, Nucleic Acids Res. 2015 January;43(Database issue):D413-22; and http://www.imgt.org/).

The antigen recognizing construct of the invention may comprise a TCR αor γ chain; and/or a TCR β or δ chain; wherein the TCR α or γ chaincomprises a CDR3 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,or 100% sequence identity to an amino acid sequence selected from SEQ IDNOs: 3, 15, 27, 39, 51, 63, 75, 87, 99, and 111, and/or wherein the TCRβ or δ chain comprises a CDR3 having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or 100% sequence identity to an amino acid sequenceselected from SEQ ID NOs: 9, 21, 33, 45, 57, 69, 81, 93, 105, and 117.

Most preferably, in some additional embodiments, wherein the disclosurerefers to antigen recognizing constructs comprising any one, two or allof the CDR1 to CDR3 regions of the herein disclosed TCR chains (seeTable 1), such antigen recognizing constructs may be preferred, whichcomprise the respective CDR sequence of the invention with not more thanthree, two, and preferably only one, modified amino acid residues. Amodified amino acid residue may be selected from an amino acidinsertion, deletion or substitution. Most preferred is that the three,two, preferably only one modified amino acid residue is the first orlast amino acid residue of the respective CDR sequence. If themodification is a substitution then it is preferable in some embodimentsthat the substitution is a conservative amino acid substitution.

If the antigen recognizing construct of the invention is composed of atleast two amino acid chains, such as a double chain TCR, or antigenbinding fragment thereof, the antigen recognizing construct maycomprises in a first polypeptide chain the amino acid sequence accordingto SEQ ID NO: 3, and in a second polypeptide chain the amino acidsequence according to SEQ ID NO: 9; or in a first polypeptide chain theamino acid sequence according to SEQ ID NO: 15, and in a secondpolypeptide chain the amino acid sequence according to SEQ ID NO: 21; orin a first polypeptide chain the amino acid sequence according to SEQ IDNO: 27, and in a second polypeptide chain the amino acid sequenceaccording to SEQ ID NO: 33; or in a first polypeptide chain the aminoacid sequence according to SEQ ID NO: 39, and in a second polypeptidechain the amino acid sequence according to SEQ ID NO: 45; or in a firstpolypeptide chain the amino acid sequence according to SEQ ID NO: 51,and in a second polypeptide chain the amino acid sequence according toSEQ ID NO: 57; or in a first polypeptide chain the amino acid sequenceaccording to SEQ ID NO: 63, and in a second polypeptide chain the aminoacid sequence according to SEQ ID NO: 69; or in a first polypeptidechain the amino acid sequence according to SEQ ID NO: 75, and in asecond polypeptide chain the amino acid sequence according to SEQ ID NO:81; or in a first polypeptide chain the amino acid sequence according toSEQ ID NO: 87, and in a second polypeptide chain the amino acid sequenceaccording to SEQ ID NO: 93; or in a first polypeptide chain the aminoacid sequence according to SEQ ID NO: 99, and in a second polypeptidechain the amino acid sequence according to SEQ ID NO: 105; or in a firstpolypeptide chain the amino acid sequence according to SEQ ID NO: 111,and in a second polypeptide chain the amino acid sequence according toSEQ ID NO: 117. Any one of the aforementioned double chain TCR, orantigen binding fragments thereof, are preferred TCR of the presentinvention. In some embodiments, the CDR3 of the double chain TCR of theinvention may be mutated. Mutations of the CDR3 sequences as providedabove preferably include a substitution, deletion, addition, orinsertion of not more than three, preferably two, and most preferablynot more than one amino acid residue. In some embodiments, the firstpolypeptide chain may be a TCR α or γ chain, and the second polypeptidechain may be a TCR β or δ chain. Preferred is the combination of an αβor γδ TCR.

The TCR, or the antigen binding fragment thereof, is in some embodimentscomposed of a TCR α and a TCR β chain, or γ and δ chain. Such a doublechain TCR comprises within each chain variable regions, and the variableregions each comprise one CDR1, one CDR2 and one CDR3 sequence. The TCRscomprises the CDR1 to CDR3 sequences as comprised in the variable chainamino acid sequence of SEQ ID NOs: 4 and 10; or 16 and 22; or 28 and 34;or 40 and 46; or 52 and 58; or 64 and 70; or 76 and 82; or 88 and 94; or100 and 106; or 112 and 118.

Some embodiments of the invention pertain to a TCR, or a fragmentthereof, composed of a TCR α and a TCR β chain, wherein said TCRcomprises the variable region sequences having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or preferably 100% sequence identity to theamino acid sequence selected from the α and β chain according to SEQ IDNOs: 4 and 10; or 16 and 22; or 28 and 34; or 40 and 46; or 52 and 58;or 64 and 70; or 76 and 82; or 88 and 94; or 100 and 106; or 112 and118.

The inventive TCRs may further comprise a constant region derived fromany suitable species, such as any mammal, e.g., human, rat, monkey,rabbit, donkey, or mouse. In an embodiment of the invention, theinventive TCRs further comprise a human constant region. In somepreferred embodiments, the constant region of the TCR of the inventionmay be slightly modified, for example, by the introduction ofheterologous sequences, preferably mouse sequences, which may increaseTCR expression and stability.

The inventive TCRs may further comprise modified T cell receptor (TCR) aor β chains, or heterodimers comprising the same, wherein in thevariable domain of said modified a or β chain, an amino acid at position44 according to the IMGT numbering is substituted with another suitableamino acid, in order to improve pairing of desired chains. According toone embodiment of said recombinant T cell receptor (TCR) heterodimer, inat least the a or β chain, the amino acid as present at position 44 inthe variable domain is substituted by one amino acid selected from thegroup consisting of Q, R, D, E, K, L, W, and V.

According to another embodiment, the inventive TCRs may further includeone of the preferred substitution pairs selected from the followinglists:

αQ44D/βQ44R; αQ44R/βQ44D; αQ44E/βQ44K; αQ44K/βQ44E; αQ44D/βQ44K;αQ44K/βQ44D; αQ44E/βQ44R; αQ44R/βQ44E; αQ44L/βQ44W; αQ44W/βQ44L;αQ44V/βQ44W; and αQ44W/βQ44V; αW44D/βQ44R; αW44R/βQ44D; αW44E/βQ44K;αW44K/βQ44E; αW44D/βQ44K; αW44K/βQ44D; αW44E/βQ44R; αW44R/βQ44E;αW44L/βQ44W; αW44/βQ44L; αW44V/βQ44W; and αW44/βQ44V; αH44D/βQ44R;αH44R/βQ44D; αH44E/βQ44K; αH44K/βQ44E; αH44D/βQ44K; αH44K/βQ44D;αH44E/βQ44R; αH44R/βQ44E; αH44L/βQ44W; αH44W/βQ44L; αH44V/βQ44W; andαH44W/βQ44V; αK44D/βQ44R; αK44R/βQ44D; αK44E/βQ44K; αK44/βQ44E;αK44D/βQ44K; αK44/βQ44D; αK44E/βQ44R; αK44R/βQ44E; αK44L/βQ44W;αK44W/βQ44L; αK44V/βQ44W; and αK44W/βQ44V; αE44D/βQ44R; αE44R/βQ44D;αE44/βQ44K; αE44K/βQ44E; αE44D/βQ44K; αE44K/βQ44D; αE44/βQ44R;αE44R/βQ44E; αE44L/βQ44W; αE44W/βQ44L; αE44V/βQ44W; and αE44W/βQ44V;αQ44D/βR44; αQ44R/βR44D; αQ44E/βR44K; αQ44K/βR44E; αQ44D/βR44K;αQ44K/βR44D; αQ44E/βR44; αQ44R/βR44E; αQ44L/βR44W; αQ44W/βR44L;αQ44V/βR44W; and αQ44W/βR44V; αW44D/βR44; αW44R/βR44D; αW44E/βR44K;αW44K/βR44E; αW44D/βR44K; αW44K/βR44D; αW44E/βR44; αW44R/βR44E;αW44L/βR44W; αW44/βR44L; αW44V/βR44W; and αW44/βR44V; αH44D/βR44;αH44R/βR44D; αH44E/βR44K; αH44K/βR44E; αH44D/βR44K; αH44K/βR44D;αH44E/βR44; αH44R/βR44E; αH44L/βR44W; αH44W/βR44L; αH44V/βR44W; andαH44W/βR44V; αK44D/βR44; αK44R/βR44D; αK44E/βR44K; αK44/βR44E;αK44D/βR44K; αK44/βR44D; αK44E/βR44; αK44R/βR44E; αK44L/βR44W;αK44W/βR44L; αK44V/βR44W; and αK44W/βR44V; αE44D/βR44; αE44R/βR44D;αE44/βR44K; αE44K/βR44E; αE44D/βR44K; αE44K/βR44D; αE44R/βR44E;αE44L/βR44W; αE44W/βR44L; αE44V/βR44W; and αE44W/βR44V.

In the above, e.g. “αQ44R/βQ44D” shall mean, for example, that, in thevariable domain of the α chain, Q44 is substituted by R, while in thevariable domain of the β chain, Q44 is substituted by D.

Some embodiments of the invention pertain to a TCR, or a fragmentthereof, composed of a TCR α and a TCR β chain, wherein said TCRcomprises the constant region having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or preferably 100% sequence identity to an amino acidsequence selected from of the α and β chain according to SEQ ID NOs: 5and 11; or 17 and 23; or 29 and 35; or 41 and 47; or 53 and 59; or 65and 71; or 77 and 83; or 89 and 95; or 101 and 107; or 113 and 119.

The TCR α or γ chain of the invention may further comprise a CDR1 havingat least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to an amino acid sequence selected from SEQ ID NOs: 1, 13, 25,37, 49, 61, 73, 85, 97, and 109; and/or a CDR2 having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID NOs: 2, 14, 26, 38, 50, 62, 74, 86, 98,and 110.

According to the invention the TCR β or δ chain may further comprise aCDR1 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%sequence identity to an amino acid sequence selected from SEQ ID NOs: 7,19, 31, 43, 55, 67, 79, 91, 103, and 115; and/or a CDR2 having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to anamino acid sequence selected from SEQ ID NOs: 8, 20, 32, 44, 56, 68, 80,92, 104, and 116.

The antigen recognizing construct may in a further embodiment comprise abinding fragment of a TCR, and wherein said binding fragment comprisesin one chain CDR1 to CDR3, optionally selected from the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NOs: 1, 2, 3; or 7,8, 9; or 13, 14, 15; or 19, 20, 21; or 25, 26, 27; or 31, 32, 33; or 37,38, 39; or 43, 44, 45; or 49, 50, 51; or 55, 56, 57; or 61, 62, 63; or67, 68, 69; or 73, 74, 75; or 79, 80, 81; or 85, 86, 87; or 91, 92, 93;or 97, 98, 99; or 103, 104, 105; or 109, 110, 111; or 115, 116, 117.

In further embodiments of the invention the antigen recognizingconstruct as described herein elsewhere is a TCR, or a fragment thereof,composed of at least one TCR α and one TCR chain sequence, wherein saidTCR α chain sequence comprises the CDR1 to CDR3 sequences having theamino acid sequences of SEQ ID NOs: 1 to 3, and said TCR β chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NOs: 7 to 9; or wherein said TCR α chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NOs: 13 to 15, and said TCR β chain sequence comprises the CDR1to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 19 to21; or wherein said TCR α chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NOs: 25 to 27, andsaid TCR β chain sequence comprises the CDR1 to CDR3 sequences havingthe amino acid sequences of SEQ ID NOs: 31 to 33; or wherein said TCR αchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NOs: 37 to 39, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NOs: 43 to 45; or wherein said TCR α chain sequence comprises theCDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 49to 51, and said TCR β chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NOs: 55 to 57; orwherein said TCR α chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NOs: 61 to 63, and said TCR βchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NOs: 67 to 69; or wherein said TCR α chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NOs: 73 to 75, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NOs: 79 to 81; or wherein said TCR α chain sequence comprises theCDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 85to 87, and said TCR β chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NOs: 91 to 93; orwherein said TCR α chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NOs: 97 to 99, and said TCR βchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NOs: 103 to 105; or wherein said TCR α chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NOs: 109 to 111, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NOs: 115 to 117.

In further embodiments of the invention the antigen recognizingconstruct as described herein before is a TCR, or a fragment thereof,comprising at least one TCR α and one TCR β chain sequence, wherein saidTCR α chain sequence comprises a variable region sequence having theamino acid sequence of SEQ ID NO: 4, and wherein said TCR β chainsequence comprises a variable region sequence having the amino acidsequence of SEQ ID NO: 10; or wherein said TCR α chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID NO: 16, and wherein said TCR β chain sequence comprises avariable region sequence having the amino acid sequence of SEQ ID NO:22; or wherein said TCR α chain sequence comprises a variable regionsequence having the amino acid sequence of SEQ ID NO: 28, and whereinsaid TCR β chain sequence comprises a variable region sequence havingthe amino acid sequence of SEQ ID NO: 34; or wherein said TCR α chainsequence comprises a variable region sequence having the amino acidsequence of SEQ ID NO: 40, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID NO: 46; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID NO: 52, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID NO: 58; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID NO: 64, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID NO: 70; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID NO: 76, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID NO: 82; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID NO: 88, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID NO: 94; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID NO: 100, andwherein said TCR chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID NO: 106; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID NO: 112, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID NO: 118.

In further embodiments of the invention the antigen recognizingconstruct as described herein before is a TCR, or a fragment thereof,further comprising a TCR constant region having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53,59, 65, 71, 77, 83, 89, 95, 101, 107, 113, and 119, preferably whereinthe TCR is composed of at least one TCR α and one TCR β chain sequence,wherein the TCR α chain sequence comprises a constant region having atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identityto an amino acid sequence selected from SEQ ID NOs: 5, 17, 29, 41, 53,65, 77, 89, 101, and 113; and wherein the TCR β chain sequence comprisesa constant region having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%,99%, or 100% sequence identity to an amino acid sequence selected fromSEQ ID NOs: 11, 23, 35, 47, 59, 71, 83, 95, 107, and 119.

Also disclosed are antigen recognizing constructs as described hereinbefore comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO: 6, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 12. The invention also provides TCRs comprising afirst TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18,and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 24. In further embodiments the invention provides antigenrecognizing constructs which are TCR and comprise a first TCR chainhaving at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 30, and a second TCRchain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 36. Infurther embodiments the invention provides antigen recognizingconstructs which are TCR and comprise a first TCR chain having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 42, and a second TCR chain having atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identityto the amino acid sequence of SEQ ID NO: 48. In further embodiments theinvention provides antigen recognizing constructs which are TCR andcomprise a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 54, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO: 60. In further embodiments the invention provides antigenrecognizing constructs which are TCR and comprise a first TCR chainhaving at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:66, and a second TCRchain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 72. Infurther embodiments the invention provides antigen recognizingconstructs which are TCR and comprise a first TCR chain having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 78, and a second TCR chain having atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identityto the amino acid sequence of SEQ ID NO: 84. In further embodiments theinvention provides antigen recognizing constructs which are TCR andcomprise a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 90, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO: 96. In further embodiments the invention provides antigenrecognizing constructs which are TCR and comprise a first TCR chainhaving at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 102, and a second TCRchain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 108. Infurther embodiments the invention provides antigen recognizingconstructs which are TCR and comprise a first TCR chain having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 114, and a second TCR chain having atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identityto the amino acid sequence of SEQ ID NO: 120.

As used herein, the term “murine” or “human,” when referring to anantigen recognizing construct, or a TCR, or any component of a TCRdescribed herein (e.g., complementarity determining region (CDR),variable region, constant region, a chain, and/or β chain), means a TCR(or component thereof), which is derived from a mouse or a humanunrearranged TCR locus, respectively.

In an embodiment of the invention, chimeric TCR are provided, whereinthe TCR chains comprise sequences from multiple species. Preferably, aTCR of the invention may comprise an α chain comprising a human variableregion of an α chain and, for example, a murine constant region of amurine TCR α chain.

In one embodiment, the TCR of the invention is a human TCR comprisinghuman variable regions according to the above embodiments and humanconstant regions.

In some embodiments the antigen recognizing construct is murinized orhumanized. These terms are used when amino acid sequences from a foreignspecies are introduced into a construct of the invention.

The TCR of the invention may be provided as a single chain TCR (scTCR).A scTCR according to the invention shall comprise in one polypeptidechain a full or partial alpha chain sequence and a full or partial betachain sequence, preferably connected via a peptide linker. A scTCR cancomprise a polypeptide of a variable region of a first TCR chain (e.g.,an alpha chain) and a polypeptide of an entire (full-length) second TCRchain (e.g., a beta chain), or vice versa. Furthermore, the scTCR canoptionally comprise one or more linkers which join the two or morepolypeptides together. The linker can be, for instance, a peptide, whichjoins together two single chains, as described herein. Also provided issuch a scTCR of the invention, which is fused to a human cytokine, suchas IL-2, IL-7 or IL-15.

The antigen recognizing construct according to the invention can also beprovided in the form of a multimeric complex, comprising at least twoscTCR molecules, wherein said scTCR molecules are each fused to at leastone biotin moiety, or other interconnecting molecule/linker, and whereinsaid scTCRs are interconnected by biotin-streptavidin interaction toallow the formation of said multimeric complex. Similar approaches knownin the art for the generation of multimeric TCR are also possible andincluded in this disclosure. Also provided are multimeric complexes of ahigher order, comprising more than two scTCR of the invention.

For the purposes of the present invention, a TCR is a moiety having atleast one TCR alpha or gamma and/or TCR beta or delta variable domain.Generally, they comprise both a TCR alpha variable domain and a TCR betavariable domain, alternatively both a TCR gamma variable domain and aTCR delta variable domain. They may be αβ/γδ heterodimers or may be insingle chain format. For use in adoptive therapy, an αβ or γδheterodimeric TCR may, for example, be transfected as full-length chainshaving both cytoplasmic and transmembrane domains. If desired, anintroduced disulfide bond between residues of the respective constantdomains may be present.

In a preferred embodiment, the antigen recognizing construct is a humanTCR, or fragment or derivative thereof. A human TCR or fragment orderivative thereof is a TCR, which comprises over 50% of thecorresponding human TCR sequence. Preferably, only a small part of theTCR sequence is of artificial origin or derived from other species. Itis known, however, that chimeric TCRs, e.g. derived from human originwith murine sequences in the constant domains, are advantageous.Particularly preferred are, therefore, TCRs in accordance with thepresent invention, which contains murine sequences in the extracellularpart of their constant domains.

Thus, it is also preferred that the inventive antigen recognizingconstruct is able to recognize its antigen in a human leucocyte antigen(HLA) dependent manner, preferably in a HLA-A*02 dependent manner. Theterm “HLA dependent manner” in the context of the present inventionmeans that the antigen recognizing construct binds to the antigen onlyin the event that the antigenic peptide is presented by said HLA.

The antigen recognizing construct in accordance with the invention inone embodiment preferably induces an immune response, preferably whereinthe immune response is characterized by the increase in interferon (IFN)γ levels.

Also provided by the invention is a polypeptide comprising a functionalportion of any of the TCRs (or functional variants thereof) describedherein, for examples, of any one of the TCRs selected from R39P1C12,R39P1F5, R40P1C2, R41P3E6, R43P3G4, R44P3B3, R44P3E7, R49P2B7, R55P1G7and R59P2A7, as provided in the example section and table 1. The term“polypeptide” as used herein includes oligopeptides and refers to asingle chain of amino acids connected by one or more peptide bonds. Withrespect to the inventive polypeptides, the functional portion can be anyportion comprising contiguous amino acids of the TCR (or functionalvariant thereof), of which it is a part, provided that the functionalportion specifically binds to the TAA antigen, preferably as disclosedherein in Table 2, and peptides A2 to A9 (SEQ ID NOs: 134-140) and T1 toT9 (SEQ ID:141-149). The term “functional portion” when used inreference to a TCR (or functional variant thereof) refers to any part orfragment of the TCR (or functional variant thereof) of the invention,which part or fragment retains the biological activity of the TCR (orfunctional variant thereof), of which it is a part (the parent TCR orparent functional variant thereof). Functional portions encompass, forexample, those parts of a TCR (or functional variant thereof) thatretain the ability to specifically bind to the TAA antigen (in an HLAdependent manner), or detect, treat, or prevent cancer, to a similarextent, the same extent, or to a higher extent, as the parent TCR (orfunctional variant thereof). In reference to the parent TCR (orfunctional variant thereof), the functional portion can comprise, forinstance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of theparent TCR variable sequences (or functional variant thereof).

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, in whichadditional amino acids are not found in the amino acid sequence of theparent TCR or functional variant thereof. Desirably, the additionalamino acids do not interfere with the biological function of thefunctional portion, e.g., specifically binding to the TAA antigens;and/or having the ability to detect cancer, treat or prevent cancer,etc. More desirably, the additional amino acids enhance the biologicalactivity, as compared to the biological activity of the parent TCR orfunctional variant thereof.

The polypeptide can comprise a functional portion of either or both ofthe α and β chains of the TCRs or functional variant thereof of theinvention, such as a functional portion comprising one of more of CDR1,CDR2, and (preferably) CDR3 of the variable region(s) of the α chainand/or β chain of a TCR or functional variant thereof of the invention.In an embodiment of the invention, the polypeptide can comprise afunctional portion comprising the amino acid sequence of SEQ ID NOs: 3,9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111,and 117 (CDR3 of the variable regions of the TCR of the invention), or acombination thereof. In an embodiment of the invention, the inventivepolypeptide can comprise, for instance, the variable region of theinventive TCR or functional variant thereof comprising a combination ofthe CDR regions set forth above. In this regard, the polypeptide cancomprise the amino acid sequence of any of SEQ ID NOs: 4, 10, 16, 22,28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, and 118(the variable regions of an α or β chain of the TCR of the invention).

In some instances, the construct of the invention may comprise one ortwo polypeptide chains comprising sequences according to any of the SEQID NOs: 1 to 120 (CDR sequences, constant and variable regions andfull-length sequences), or functional fragments thereof, and furthercomprise(s) other amino acid sequences, e.g., an amino acid sequenceencoding an immunoglobulin or a portion thereof, then the inventiveprotein can be a fusion protein. In this regard, the invention alsoprovides a fusion protein comprising at least one of the inventivepolypeptides described herein along with at least one other polypeptide.The other polypeptide can exist as a separate polypeptide of the fusionprotein, or can exist as a polypeptide, which is expressed in frame (intandem) with one of the inventive polypeptides described herein. Theother polypeptide may include any peptidic or proteinaceous molecule, ora portion thereof, including, but not limited to an immunoglobulin, CD3,CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CD1a, CD1b, CD1c, CD1d,etc.

The fusion protein can comprise one or more copies of the inventivepolypeptide and/or one or more copies of the other polypeptide. Forinstance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copiesof the inventive polypeptide and/or of the other polypeptide. Suitablemethods of making fusion proteins are known in the art, and include, forexample, recombinant methods. In some embodiments of the invention, theTCRs (and functional portions and functional variants thereof),polypeptides, and proteins of the invention may be expressed as a singleprotein comprising a linker peptide linking the α chain and the β chain,and linking the γ chain and the δ chain. In this regard, the TCRs (andfunctional variants and functional portions thereof), polypeptides, andproteins of the invention comprising the amino acid sequences of thevariable regions of the TCR of the invention and may further comprise alinker peptide. The linker peptide may advantageously facilitate theexpression of a recombinant TCR (including functional portions andfunctional variants thereof), polypeptide, and/or protein in a hostcell. The linker peptide may comprise any suitable amino acid sequence.Linker sequences for single chain TCR constructs are well known in theart. Such a single chain construct may further comprise one, or two,constant domain sequences. Upon expression of the construct includingthe linker peptide by a host cell, the linker peptide may also becleaved, resulting in separated α and β chains, and separated γ and δchain.

As already mentioned above, the binding functionality of the TCR of theinvention may be provided in the framework of an antibody. For example,CDR sequences of the TCR of the invention, possibly including additional3, 2 or 1 N and/or C terminal framework residues, may be directlygrafted into an antibody variable heavy/light chain sequence. The term“antibody” in its various grammatical forms is used herein to refer toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain anantigen-binding site or a paratope. Such molecules are also referred toas “antigen binding fragments” of immunoglobulin molecules. Theinvention further provides an antibody, or antigen binding portionthereof, which specifically binds to the antigens described herein. Theantibody can be any type of immunoglobulin that is known in the art. Forinstance, the antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG,IgM, etc. The antibody can be monoclonal or polyclonal. The antibody canbe a naturally-occurring antibody, e.g., an antibody isolated and/orpurified from a mammal, e.g., mouse, rabbit, goat, horse, chicken,hamster, human, etc. Alternatively, the antibody can be agenetically-engineered antibody, e.g., a humanized antibody or achimeric antibody. The antibody can be in monomeric or polymeric form.

The term “antibody” includes, but is not limited to, geneticallyengineered or otherwise modified forms of immunoglobulins, such asintrabodies, chimeric antibodies, fully human antibodies, humanizedantibodies (e.g. generated by “CDR-grafting”), antibody fragments, andheteroconjugate antibodies (e.g., bispecific antibodies, diabodies,triabodies, tetra-bodies, etc.). The term “antibody” includescys-diabodies and minibodies. Thus, each and every embodiment providedherein in regard to “antibodies”, or “antibody like constructs” is alsoenvisioned as, bispecific antibodies, diabodies, scFv fragments,chimeric antibody receptor (CAR) constructs, diabody and/or minibodyembodiments, unless explicitly denoted otherwise. The term “antibody”includes a polypeptide of the immunoglobulin family or a polypeptidecomprising fragments of an immunoglobulin that is capable ofnon-covalently, reversibly, and in a specific manner binding acorresponding antigen, preferably the TAA of the invention, as disclosedherein. An exemplary antibody structural unit comprises a tetramer. Insome embodiments, a full-length antibody can be composed of twoidentical pairs of polypeptide chains, each pair having one “light” andone “heavy” chain (connected through a disulfide bond). Antibodystructure and isotypes are well known to the skilled artisan (forexample from Janeway's Immunobiology, 9th edition, 2016).

The recognized immunoglobulin genes of mammals include the kappa,lambda, alpha, gamma, delta, epsilon, and mu constant region genes, aswell as the myriad immunoglobulin variable region genes (for moreinformation on immunoglobulin genes see the international ImMunoGeneTicsinformation System®, Lefranc M-P et al, Nucleic Acids Res. 2015 January;43(Database issue):D413-22; and http://www.imgt.org/). For full-lengthchains, the light chains are classified as either kappa or lambda. Forfull-length chains, the heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD, and IgE, respectively. The N-terminus of each chaindefines a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The terms variable lightchain (VL) and variable heavy chain (VH) refer to these regions of lightand heavy chains respectively. As used in this invention, an “antibody”encompasses all variations of antibody and fragments thereof. Thus,within the scope of this concept are full length antibodies, chimericantibodies, humanized antibodies, single chain antibodies (scFv), Fab,Fab′, and multimeric versions of these fragments (e.g., F(ab′)2) withthe same, essentially the same or similar binding specificity. In someembodiments, the anti-body binds specifically to a peptide TAA of theinvention. Preferred antigen recognizing constructs according to theinvention include an antibody heavy chain, preferably the variabledomain thereof, or an antigen binding fragment thereof, and/or anantibody light chain, preferably the variable domain thereof, or anantigen binding fragment thereof. Similarly, disulfide-stabilizedvariable region fragments (dsFv) can be prepared by recombinant DNAtechnology, antibody fragments of the invention, however, are notlimited to these exemplary types of antibody fragments. Also, theantibody, or antigen binding portion thereof, can be modified tocomprise a detectable label, such as, for instance, a radioisotope, afluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin(PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase),and element particles (e.g., gold particles). In some instances, the TCRCDR3 sequence may be slightly modified, but preferably by not more than3 amino acid residues, preferably only two and most preferably only oneamino acid position, as compared to the CDR3 sequences provided in SEQID NO of the CDR3 in table 1. Preferably, the antibodies comprise theCDR3, preferably all of CDR1 to CDR3 regions in the combination, asindicated for the TCR of the invention in table 1, in each caseindependently, optionally with not more than three or two, preferablyone, amino acid substitution(s), insertion(s) and/or deletion(s)compared to these sequences.

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Kohler andMilstein, Eur. J. Immunol, 5, 51 1-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 8 Ed., Garland Publishing, New York, N.Y.(2011)). Alternatively, other methods, such as EBV-hybridoma methods(Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), andRoder et al, Methods Enzymol, 121, 140-67 (1986)), and bacteriophagevector expression systems (see, e.g., Huse et al., Science, 246, 1275-81(1989)) are known in the art. Further, methods of producing antibodiesin non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,5,569,825, and 5,714,352, and U.S. Patent Application Publication No.2002/0197266.

Some embodiments of the invention also pertain to TCRs, or functionalfragments and polypeptides thereof, which are soluble TCRs. As usedherein, the term “soluble T-cell receptor” refers to heterodimerictruncated variants of native TCRs, which comprise extracellular portionsof the TCR α-chain and β-chain, for example linked by a disulfide bond,but which lack the transmembrane and cytosolic domains of the nativeprotein. The terms “soluble T-cell receptor α-chain sequence and solubleT-cell receptor β-chain sequence” refer to TCR α-chain and β-chainsequences that lack the transmembrane and cytosolic domains. Thesequence (amino acid or nucleic acid) of the soluble TCR α-chain andβ-chains may be identical to the corresponding sequences in a native TCRor may comprise variant soluble TCR α-chain and β-chain sequences, ascompared to the corresponding native TCR sequences. The term “solubleT-cell receptor” as used herein encompasses soluble TCRs with variant ornon-variant soluble TCR α-chain and β-chain sequences. The variationsmay be in the variable or constant regions of the soluble TCR α-chainand β-chain sequences and can include, but are not limited to, aminoacid deletion, insertion, substitution mutations as well as changes tothe nucleic acid sequence, which do not alter the amino acid sequence.Soluble TCR of the invention in any case retain the bindingfunctionality of their parent molecules.

The above problem is further solved by a nucleic acid encoding for anantigen recognizing construct of the invention, or any of theaforementioned protein or polypeptide constructs. The nucleic acidpreferably (a) has a strand encoding for an antigen recognizingconstruct according to the invention; (b) has a strand complementary tothe strand in (a); or (c) has a strand that hybridizes under stringentconditions with a molecule as described in (a) or (b). Stringentconditions are known to the person of skill in the art, specificallyfrom Sambrook et al, “Molecular Cloning”. In addition to that, thenucleic acid optionally has further sequences, which are necessary forexpressing the nucleic acid sequence corresponding to the protein,specifically for expression in a mammalian/human cell. The nucleic acidused can be contained in a vector suitable for allowing expression ofthe nucleic acid sequence corresponding to the peptide in a cell.However, the nucleic acids can also be used to transform anantigen-presenting cell, which may not be restricted to classicalantigen-presenting cells, such as dendritic cells, in such a way thatthey themselves produce the corresponding proteins on their cellularsurface.

In some embodiments, the polypeptides of the antigen recognizingconstructs can be encoded by nucleic acids and expressed in vivo or invitro. Thus, in some embodiments, a nucleic acid encoding an antigenrecognizing construct is provided. In some embodiments, the nucleic acidencodes one part or monomer of an antigen recognizing construct of theinvention (for example one of two chains of a TCR of the invention),and/or another nucleic acid encodes another part or monomer of anantigen recognizing construct of the invention (for example the other oftwo chains of the TCR). In some embodiments, the nucleic acid encodestwo or more antigen recognizing construct polypeptide chains, forexample, at least 2 TCR chains. Nucleic acids encoding multiple antigenrecognizing construct chains can include nucleic acid cleavage sitesbetween at least two chain sequences, can encode transcription ortranslation start site between two or more chains sequences, and/or canencode proteolytic target sites between two or more antigen recognizingconstruct chains.

By “nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and can contain a natural, nonnatural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication. The nucleic acid can compriseany nucleotide sequence, which encodes any of the TCRs, polypeptides, orproteins, or functional portions or functional variants thereofdescribed herein.

Furthermore, the invention provides a vector comprising a nucleic acidin accordance to the invention as described above. Desirably, the vectoris an expression vector or a recombinant expression vector. The term“recombinant expression vector” refers in context of the presentinvention to a nucleic acid construct that allows for the expression ofan mRNA, protein or polypeptide in a suitable host cell. The recombinantexpression vector of the invention can be any suitable recombinantexpression vector, and can be used to transform or transfect anysuitable host. Suitable vectors include those designed for propagationand expansion or for expression or both, such as plasmids and viruses.Examples of animal expression vectors include pEUK-Cl, pMAM, andpMAMneo. Preferably, the recombinant expression vector is a viralvector, e.g., a retroviral vector. The recombinant expression vectorcomprises regulatory sequences, such as transcription and translationinitiation and termination codons, which are specific to the type ofhost cell (e.g., bacterium, fungus, plant, or animal), into which thevector is to be introduced and in which the expression of the nucleicacid of the invention may be performed. Furthermore, the vector of theinvention may include one or more marker genes, which allow forselection of transformed or transfected hosts. The recombinantexpression vector can comprise a native or normative promoter operablylinked to the nucleotide sequence encoding the constructs of theinvention, or to the nucleotide sequence, which is complementary to orwhich hybridizes to the nucleotide sequence encoding the constructs ofthe invention. The selections of promoters include, e.g., strong, weak,inducible, tissue-specific and developmentalspecific promoters. Thepromoter can be a non-viral promoter or a viral promoter. The inventiverecombinant expression vectors can be designed for either transientexpression, for stable expression, or for both. Also, the recombinantexpression vectors can be made for constitutive expression or forinducible expression.

The invention also pertains to a host cell comprising an antigenrecognizing construct in accordance with the invention. Specifically,the host cell of the invention comprises a nucleic acid, or a vector asdescribed herein above. The host cell can be a eukaryotic cell, e.g.,plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g.,bacteria or protozoa. The host cell can be a cultured cell or a primarycell, i.e., isolated directly from an organism, e.g., a human. The hostcell can be an adherent cell or a suspended cell, i.e., a cell thatgrows in suspension. For purposes of producing a recombinant TCR,polypeptide, or protein, the host cell is preferably a mammalian cell.Most preferably, the host cell is a human cell. While the host cell canbe of any cell type, can originate from any type of tissue, and can beof any developmental stage, the host cell preferably is a peripheralblood leukocyte (PBL) or a peripheral blood mononuclear cell (PBMC).More preferably, the host cell is a T cell. The T cell can be any Tcell, such as a cultured T cell, e.g., a primary T cell, or a T cellfrom a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cellobtained from a mammal, preferably a T cell or T cell precursor from ahuman patient. If obtained from a mammal, the T cell can be obtainedfrom numerous sources, including but not limited to blood, bone marrow,lymph node, the thymus, or other tissues or fluids. T cells can also beenriched for or purified. Preferably, the T cell is a human T cell. Morepreferably, the T cell is a T cell isolated from a human. The T cell canbe any type of T cell and can be of any developmental stage, includingbut not limited to, CD4-positive and/or CD8-positive, CD4-positivehelper T cells, e.g., Th1 and Th2 cells, CD8-positive T cells (e.g.,cytotoxic T cells), tumor infiltrating cells (TILs), memory T cells,naive T cells, and the like. Preferably, the T cell is a CD8-positive Tcell or a CD4-positive T cell.

Preferably, the host cell of the invention is a lymphocyte, preferably,a T lymphocyte, such as a CD4-positive or CD8-positive T-cell. The hostcell furthermore preferably is a tumor reactive T cell specific for TAAexpressing tumor cells.

The objective of the invention is also solved by a method ofmanufacturing a TAA specific antigen recognizing construct, or of a TAAspecific antigen recognizing construct expressing cell line, comprising

-   a. Providing a suitable host cell,-   b. Providing a genetic construct comprising a coding sequence    encoding for an antigen recognizing construct according to the    herein disclosed invention,-   c. Introducing into said suitable host cell said genetic construct,    and-   d. Expressing said genetic construct by said suitable host cell.

The method may further comprise a step of cell surface presentation ofsaid antigen recognizing construct on said suitable host cell.

In other preferred embodiments, the genetic construct is an expressionconstruct comprising a promoter sequence operably linked to said codingsequence.

Preferably, said antigen recognizing construct is of mammalian origin,preferably of human origin. The preferred suitable host cell for use inthe method of the invention is a mammalian cell, such as a human cell,in particular a human T lymphocyte. T cells for use in the invention aredescribed in detail herein above.

Also encompassed by the invention are embodiments, wherein said antigenrecognizing construct is a modified TCR, wherein said modification isthe addition of functional domains, such as a label or a therapeuticallyactive substance. Furthermore, encompassed are TCR having alternativedomains, such as an alternative membrane anchor domain instead of theendogenous transmembrane region.

Desirably, the transfection system for introducing the genetic constructinto said suitable host cell is a retroviral vector system. Such systemsare well known to the skilled artisan.

Also comprised by the present invention is in one embodiment theadditional method step of isolation and purification of the antigenrecognizing construct from the cell and, optionally, the reconstitutionof the translated antigen recognizing construct-fragments in a T-cell.

In an alternative aspect of the invention a T-cell is provided obtainedor obtainable by a method for the production of a T cell receptor (TCR),which is specific for tumorous cells and has high avidity as describedherein above. Such a T cell is depending on the host cell used in themethod of the invention, for example, a human or non-human T-cell,preferably a human TCR.

The term “isolated” as used herein in the context of a polypeptide, suchas an antigen recognizing construct (an example of which could be anantibody), refers to a polypeptide that is purified from proteins orpolypeptides or other contaminants that would interfere with itstherapeutic, diagnostic, prophylactic, research or other use. An antigenrecognizing construct according to the invention may be a recombinant,synthetic or modified (non-natural) antigen binding construct. The term“isolated” as used herein in the context of a nucleic acid or cellsrefers to a nucleic acid or cells that is/are purified from DNA, RNA,proteins or polypeptides or other contaminants (such as other cells)that would interfere with its therapeutic, diagnostic, prophylactic,research or other use, or it refers to a recombinant, synthetic ormodified (non-natural) nucleic acid. In this context, a “recombinant”protein/polypeptide or nucleic acid is one made using recombinanttechniques. Methods and techniques for the production of recombinantnucleic acids and proteins are well known in the art.

Treatment Methods and Diseases

One further aspect of the present invention relates to the hereindisclosed antigen recognizing constructs, nucleic acids, vectors,pharmaceutical compositions and/or host cell for use in medicine. Theuse in medicine in one preferred embodiment includes the use in thediagnosis, prevention and/or treatment of a tumor disease, such as amalignant or benign tumor disease. The tumor disease is, for example, atumor disease characterized by the expression of the TAA, in a cancer ortumor cell of said tumor disease.

With respect to the above mentioned medical applications of the antigenrecognizing constructs and other materials derived therefrom, pertainingthereto or encoding the same, in accordance of the present disclosure,the to be treated and/or to be diagnosed diseases can be anyproliferative disorder, preferably characterized by the expression ofthe TAA or TAA epitope sequence of the invention, for example anycancer, including any of acute lymphocytic cancer, acute myeloidleukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breastcancer, cancer of the anus, anal canal, or anorectum, cancer of the eye,cancer of the intrahepatic bile duct, cancer of the joints, cancer ofthe neck, gallbladder, or pleura, cancer of the nose, nasal cavity, ormiddle ear, cancer of the oral cavity, cancer of the vagina, cancer ofthe vulva, chronic lymphocytic leukemia, chronic myeloid cancer, coloncancer, esophageal cancer, cervical cancer, gastrointestinal carcinoidtumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma,cancer of the oropharynx, ovarian cancer, cancer of the penis,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, stomach cancer, testicular cancer,thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladdercancer. A preferred cancer is cancer is cancer of the uterine cervix,oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis. Aparticularly preferred cancer is a TAA positive cancer, includingpreferably lymphoma.

The constructs, proteins, TCRs antibodies, polypeptides and nucleicacids of the invention are in particular for use in immune therapy,preferably, in adoptive T cell therapy. The administration of thecompounds of the invention can, for example, involve the infusion of Tcells of the invention into said patient. Preferably, such T cells areautologous T cells of the patient and in vitro transduced with a nucleicacid or antigen recognizing construct of the present invention.

The inventive antigen recognizing constructs, TCRs, polypeptides,proteins (including functional variants thereof), nucleic acids,recombinant expression vectors, host cells (including populationsthereof), and antibodies (including antigen binding portions thereof),all of which are collectively referred to as “inventive TCR materials”hereinafter, can be formulated into a composition, such as apharmaceutical composition. In this regard, the invention provides apharmaceutical composition comprising any of the antigen recognizingconstructs, TCRs, polypeptides, proteins, functional portions,functional variants, nucleic acids, expression vectors, host cells(including populations thereof), and antibodies (including antigenbinding portions thereof) described herein, and a pharmaceuticallyacceptable carrier, excipient and/or stabilizer. The inventivepharmaceutical compositions containing any of the inventive TCRmaterials can comprise more than one inventive TCR material, e.g., apolypeptide and a nucleic acid, or two or more different TCRs (includingfunctional portions and functional variants thereof). Alternatively, thepharmaceutical composition can comprise an inventive TCR material incombination with another pharmaceutically active agent(s) or drug(s),such as chemotherapeutic agents, e.g., asparaginase, busulfan,carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil,gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab,vinblastine, vincristine, etc. Preferably, the carrier is apharmaceutically acceptable carrier. With respect to pharmaceuticalcompositions, the carrier can be any of those conventionally used forthe particular inventive TCR material under consideration. Suchpharmaceutically acceptable carriers are well-known to those skilled inthe art and are readily available to the public. It is preferred thatthe pharmaceutically acceptable carrier be one, which has no detrimentalside effects or toxicity under the conditions of use.

Thus also provided is a pharmaceutical composition, comprising any ofthe herein described products of the invention and TCR materials of theinvention, specifically any proteins, nucleic acids or host cells. In apreferred embodiment the pharmaceutical composition is for immunetherapy, preferably adoptive cell therapy.

Preferably, the inventive TCR material is administered by injection,e.g., intravenously. When the inventive TCR material is a host cellexpressing the inventive TCR (or functional variant thereof), thepharmaceutically acceptable carrier for the cells for injection mayinclude any isotonic carrier such as, for example, normal saline (about0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott,Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5%dextrose in water, or Ringer's lactate. In an embodiment, thepharmaceutically acceptable carrier is supplemented with human serumalbumen.

For purposes of the invention, the amount or dose (e.g., numbers ofcells when the inventive TCR material is one or more cells) of theinventive TCR material administered may be sufficient to affect, e.g., atherapeutic or prophylactic response, in the subject or animal over areasonable time frame. For example, the dose of the inventive TCRmaterial should be sufficient to bind to a cancer antigen, or detect,treat or prevent cancer in a period of from about 2 hours or longer,e.g., 12 to 24 or more hours, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular inventive TCR materialand the condition of the animal (e.g., human), as well as the bodyweight of the animal (e.g., human) to be treated.

It is contemplated that the inventive pharmaceutical compositions,antigen recognizing constructs, TCRs (including functional variantsthereof), polypeptides, proteins, nucleic acids, recombinant expressionvectors, host cells, or populations of cells can be used in methods oftreating or preventing cancer, or TAA-positive premalignancy. Theinventive TCRs (and functional variants thereof) are believed to bindspecifically to the TAA of the invention, such that the TCR (or relatedinventive polypeptide or protein and functional variants thereof), whenexpressed by or on a cell, such as a T cell, is able to mediate animmune response against a target cell expressing the TAA of theinvention, preferably presenting TAA peptides via MHC I or II on thesurface of said target cell. In this regard, the invention provides amethod of treating or preventing a condition, in particular cancer, in amammal, comprising administering to the mammal any of the pharmaceuticalcompositions, antigen recognizing constructs, in particular TCRs (andfunctional variants thereof), polypeptides, or proteins describedherein, any nucleic acid or recombinant expression vector comprising anucleotide sequence encoding any of the TCRs (and functional variantsthereof), polypeptides, proteins described herein, or any host cell orpopulation of cells comprising a nucleic acid or recombinant vector,which encodes any of the constructs of the invention (and functionalvariants thereof), polypeptides, or proteins described herein, in anamount effective to treat or prevent the condition in the mammal,wherein the condition is preferably cancer, such as a cancer expressingthe TAA of the invention.

Examples of pharmaceutically acceptable carriers or diluents useful inthe present invention include stabilizers such as SPGA, carbohydrates(e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteinssuch as albumin or casein, protein containing agents such as bovineserum or skimmed milk and buffers (e.g. phosphate buffer).

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof a condition in a mammal. Furthermore, the treatment or preventionprovided by the inventive method can include treatment or prevention ofone or more conditions or symptoms of the condition, e.g., cancer, beingtreated or prevented. For example, treatment or prevention can includepromoting the regression of a tumor. Also, for purposes herein,“prevention” can encompass delaying the onset of the condition, or asymptom or condition thereof.

The present invention also relates to a method of treating cancercomprising administering a TCR, a nucleic acid, or a host cell of thepresent description in combination with at least one chemotherapeuticagent and/or radiation therapy.

Another aspect of the invention further pertains to a method fordetecting a TAA protein, or a complex of MHC and the TAA protein(protein epitope of the TAA), in a (biological) sample—such as oneobtained from a subject or patient—comprising contacting the sample withan antigen recognizing construct specifically binding to said TAApeptide, or to the TAA peptide/MHC complex, and detecting the bindingbetween said antigen recognizing construct and said TAA peptide, or tothe TAA peptide/MHC complex. In some embodiments, the antigenrecognizing construct is a TCR or antibody, or similar constructs, orpreferably the antigen recognizing construct according to the hereindescribed invention. In some embodiments, the (biological) sample is asample of a tumor or a cancer (such as one of those described elsewhereherein) for example a sample comprising tumor or cancer cells.

Also provided is a method of treating cancer in a subject in needthereof, comprising:

-   -   a) isolating a cell from said subject;    -   b) transforming the cell with at least one vector encoding an        antigen recognizing construct of the present invention to        produce a transformed cell;    -   c) expanding the transformed cell to produce a plurality of        transformed cells; and    -   d) administering the plurality of transformed cells to said        subject.

Also provided is a method of treating cancer in a subject in needthereof, comprising:

-   -   a) isolating a cell from a healthy donor;    -   b) transforming the cell with a vector encoding an antigen        recognizing construct of the present invention to produce a        transformed cell;    -   c) expanding the transformed cell to produce a plurality of        transformed cells; and    -   d) administering the plurality of transformed cells to said        subject.

Also provided is a method of detecting cancer in a biological samplecomprising:

-   -   a) contacting the biological sample with an antigen recognizing        construct of the present description;    -   b) detecting binding of the antigen recognizing construct to the        biological sample.

In some embodiments, the method of detecting cancer is carried out invitro, in vivo or in situ.

Also provided is a method of detecting the presence of a condition in amammal. The method comprises (i) contacting a sample comprising one ormore cells from the mammal with any of the inventive TCRs (andfunctional variants thereof), polypeptides, proteins, nucleic acids,recombinant expression vectors, host cells, populations of cells,antibodies, or antigen binding portions thereof, or pharmaceuticalcompositions described herein, thereby forming a complex, and detectingthe complex, wherein detection of the complex is indicative of thepresence of the condition in the mammal, wherein the condition iscancer, such as a TAA expressing malignancy.

With respect to the inventive method of detecting a condition in amammal, the sample of cells can be a sample comprising whole cells,lysates thereof, or a fraction of the whole cell lysates, e.g., anuclear or cytoplasmic fraction, a whole protein fraction, or a nucleicacid fraction.

For purposes of the inventive detecting method, the contacting can takeplace in vitro or in vivo with respect to the mammal. Preferably, thecontacting is in vitro.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the inventive antigen recognizingconstructs (and functional variants thereof), polypeptides, proteins,nucleic acids, recombinant expression vectors, host cells, populationsof cells, or antibodies or TCRs, or antigen binding portions thereof,described herein, can be labeled with a detectable label such as, forinstance, a radioisotope, a fluorophore (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), and element particles (e.g., goldparticles).

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the mammal. Preferably, the cells are autologous to themammal.

With respect to the above mentioned medical applications of the TCRmaterial of the invention, the to be treated and/or diagnosed cancer canbe any cancer, including any of acute lymphocytic cancer, acute myeloidleukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breastcancer, cancer of the anus, anal canal, or anorectum, cancer of the eye,cancer of the intrahepatic bile duct, cancer of the joints, cancer ofthe neck, gallbladder, or pleura, cancer of the nose, nasal cavity, ormiddle ear, cancer of the oral cavity, cancer of the vagina, cancer ofthe vulva, chronic lymphocytic leukemia, chronic myeloid cancer, coloncancer, esophageal cancer, cervical cancer, gastrointestinal carcinoidtumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma,cancer of the oropharynx, ovarian cancer, cancer of the penis,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, stomach cancer, testicular cancer,thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladdercancer. A preferred cancer is cancer of the uterine cervix, oropharynx,anus, anal canal, anorectum, vagina, vulva, or penis. A particularlypreferred cancer is a TAA positive cancer, such as a Spink2 positivelymphoma.

In general, the invention provides a method for treating a subjectsuffering from a tumor or tumor disease comprising the administration ofthe antigen recognizing constructs, nucleic acids, vectors,pharmaceutical compositions and/or host cell as disclosed by the presentinvention. Preferably the subject is a subject in need of such atreatment. The subject in preferred embodiments is a mammalian subject,preferably a human patient, suffering from a tumor or tumor disease,which is TAA-positive.

In view of the disclosure herein it will be appreciated that theinvention furthermore pertains to the following items:

Item 1: An antigen recognizing construct comprising at least onecomplementary determining region (CDR) 3 having at least 50% sequenceidentity to an amino acid sequence selected from SEQ ID NOs: 3, 9, 15,21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, and117.

Item 2: The antigen recognizing construct according to item 1, whereinsaid antigen recognizing construct is capable of specifically and/orselectively binding to a TAA of the invention antigenic peptide.

Item 3: The antigen recognizing construct according to item 1 or 2,wherein the antigen recognizing construct is an antibody, or derivativeor fragment thereof, or a T cell receptor (TCR), or a derivative orfragment thereof.

Item 4: The antigen recognizing construct according to any one of items1 to 3, wherein said antigen recognizing construct binds to a humanleucocyte antigen (HLA) presented TAA antigenic peptide, wherein saidHLA is optionally type A2.

Item 5: The antigen recognizing construct according to any one of items1 to 4, wherein the construct specifically and/or selectively binds toan epitope having the amino acid sequence selected from SEQ ID NOs: 133to 158.

Item 6: The antigen recognizing construct according to any one of items1 to 5, wherein the construct is an α/β-TCR, or fragment or derivativethereof, or the construct is a γ/δ-TCR, or a fragment or derivativethereof.

Item 7: The antigen recognizing construct according to any one of items1 to 6, characterized in that the construct is of human origin andspecifically and/or selectively recognizes a TAA antigenic peptide.

Item 8: The antigen recognizing construct according to any one of items1 to 7, wherein said antigen recognizing construct is capable ofinducing an immune response in a subject, optionally wherein the immuneresponse is characterized by an increase in interferon (IFN) γ levels.

Item 9: The antigen recognizing construct according to any one of items1 to 8, comprising a TCR α or γ chain; and/or a TCR β or δ chain;wherein the TCR α or γ chain comprises a CDR3 having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID NOs: 3, 15, 27, 39, 51, 63, 75, 87, 99,and 111, and/or wherein the TCR β or δ chain comprises a CDR3 having atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identityto an amino acid sequence selected from SEQ ID NOs: 9, 21, 33, 45, 57,69, 81, 93, 105, and 117.

Item 10: The antigen recognizing construct according to item 9, whereinthe TCR α or γ chain further comprises a CDR1 having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID NOs: 1, 13, 25, 37, 49, 61, 73, 85, 97,and 109; and/or a CDR2 having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 100% sequence identity to an amino acid sequence selectedfrom SEQ ID NOs: 2, 14, 26, 38, 50, 62, 74, 86, 98, and 110.

Item 11: The antigen recognizing construct according to item 9 or 10,wherein the TCR β or 6 chain further comprises a CDR1 having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to anamino acid sequence selected from SEQ ID NOs: 7, 19, 31, 43, 55, 67, 79,91, 103, and 115; and/or a CDR2 having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or 100% sequence identity to an amino acid sequenceselected from SEQ ID NOs: 8, 20, 32, 44, 56, 68, 80, 92, 104, and 116.

Item 12: The antigen recognizing construct according to any of items 1to 11, comprising a TCR variable chain region having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52,58, 64, 70, 76, 82, 88, 94, 100, 106, 112, and 118.

Item 13: The antigen recognizing construct according to any of items 1to 12, wherein the construct is humanized, chimerized and/or murinized.

Item 14: The antigen recognizing construct according to any of items 1to 13, comprising a binding fragment of a TCR, and wherein said bindingfragment comprises CDR1 to CDR3 optionally selected from the CDR1 toCDR3 sequences having the amino acid sequences of SEQ ID NOs: 1, 2, 3;or 7, 8, 9; or 13, 14, 15; or 19, 20, 21; or 25, 26, 27; or 31, 32, 33;or 37, 38, 39; or 43, 44, 45; or 49, 50, 51; or 55, 56, 57; or 61, 62,63; or 67, 68, 69; or 73, 74, 75; or 79, 80, 81; or 85, 86, 87; or 91,92, 93; or 97, 98, 99; or 103, 104, 105; or 109, 110, 111; or 115, 116,117.

Item 15: The antigen recognizing construct according to any of items 1to 14, wherein the construct is a TCR, or a fragment thereof, composedof at least one TCR α and one TCR β chain sequence, wherein said TCR αchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NOs: 1 to 3, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NOs: 7 to 9; or wherein said TCR α chain sequence comprises theCDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 13to 15, and said TCR β chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NOs: 19 to 21; orwherein said TCR α chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NOs: 25 to 27, and said TCR βchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NOs: 31 to 33; or wherein said TCR α chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NOs: 37 to 39, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NOs: 43 to 45; or wherein said TCR α chain sequence comprises theCDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 49to 51, and said TCR β chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NOs: 55 to 57; orwherein said TCR α chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NOs: 61 to 63, and said TCR βchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NOs: 67 to 69; or wherein said TCR α chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NOs: 73 to 75, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NOs: 79 to 81; or wherein said TCR α chain sequence comprises theCDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 85to 87, and said TCR β chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NOs: 91 to 93; orwherein said TCR α chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NOs: 97 to 99, and said TCR βchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NOs: 103 to 105; or wherein said TCR α chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NOs: 109 to 111, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NOs: 115 to 117.

Item 16: The antigen recognizing construct according to any of items 1to 15, wherein the construct is a TCR, or a fragment thereof, comprisingat least one TCR α and one TCR β chain sequence, wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID NO: 4, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID NO: 10; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID NO: 16, andwherein said TCR I chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID NO: 22; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID NO: 28, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID NO: 34; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID NO: 40, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID NO: 46; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID NO: 52, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID NO: 58; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID NO: 64, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID NO: 70; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID NO: 76, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID NO: 82; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID NO: 88, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID NO: 94; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID NO: 100, and wherein said TCR chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID NO: 106; or wherein said TCR α chain sequence comprises avariable region sequence having the amino acid sequence of SEQ ID NO:112, and wherein said TCR β chain sequence comprises a variable regionsequence having the amino acid sequence of SEQ ID NO: 118.

Item 17: The antigen recognizing construct according to any of items 1to 16, wherein the construct is a TCR, or a fragment thereof, furthercomprising a TCR constant region having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequenceselected from SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71,77, 83, 89, 95, 101, 107, 113, and 119, preferably wherein the TCR iscomposed of at least one TCR α and one TCR β chain sequence, wherein theTCR α chain sequence comprises a constant region having at least 50%,60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an aminoacid sequence selected from SEQ ID NOs: 5, 17, 29, 41, 53, 65, 77, 89,101, and 113; and wherein the TCR β chain sequence comprises a constantregion having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%sequence identity to an amino acid sequence selected from SEQ ID NOs:11, 23, 35, 47, 59, 71, 83, 95, 107, and 119.

Item 18: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO: 6, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 12.

Item 19: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO: 18, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 24.

Item 20: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO: 30, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 36.

Item 20b: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO: 42, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 48.

Item 20c: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO: 54, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 60.

Item 20d: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO:66, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 72.

Item 20e: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO: 78, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 84.

Item 20f: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO: 90, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 96.

Item 20g: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO: 102, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 108.

Item 20h: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO: 114, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 120.

Item 21: A nucleic acid encoding for an antigen recognizing constructaccording to any one of items 1 to 20.

Item 22: A vector comprising a nucleic acid according to item 21.

Item 23: A host cell comprising an antigen recognizing constructaccording to any one of items 1 to 20, or a nucleic acid according toitem 21, or a vector according to item 22.

Item 24: The host cell according to item 23, wherein the cell is alymphocyte, preferably a T lymphocyte or T lymphocyte progenitor, morepreferably a CD4 or CD8 positive T-cell.

Item 25: A pharmaceutical composition comprising the antigen recognizingconstruct according to any of items 1 to 20, or the nucleic acidaccording to item 21, or the vector according to item 22, or the hostcell according to item 23 or 24, and a pharmaceutical acceptablecarrier, stabilizer and/or excipient.

Item 26: The antigen recognizing construct according to any one of items1 to 20, or a nucleic acid according to item 21, or a vector accordingto item 22, or a host cell according to item 23 or 24, or thepharmaceutical composition according to item 25, for use in medicine.

Item 27: The antigen recognizing construct, or the nucleic acid, or thevector, or the host cell, or the pharmaceutical composition, for useaccording to item 26, for use in the diagnosis, prevention, and/ortreatment of a proliferative disease, wherein the disease comprises amalignant or benign tumor disease.

Item 28: The antigen recognizing construct, or the nucleic acid, or thevector, or the host cell, or the pharmaceutical composition, for useaccording to item 27, wherein the tumor disease is characterized by theexpression of TAA in a tumor cell of the tumor disease.

Item 29: The antigen recognizing construct, or the nucleic acid, or thevector, or the host cell, or the pharmaceutical composition, for useaccording to any one of items 26 to 28, wherein the use in medicine is ause in immune therapy optionally comprising an adoptive cell transfer,wherein the immune therapy comprises adoptive autologous or heterologousT-cell therapy.

Item 30: A method of manufacturing a TAA specific antigen recognizingconstruct expressing cell line, comprising

a., providing a suitable host cell,b., providing a genetic construct comprising a coding sequence encodingthe antigen recognizing construct according to any of items 1 to 20,c., introducing into said suitable host cell said genetic construct,d., expressing said genetic construct by said suitable host cell.

Item 31: The method according to item 30, further comprising cellsurface presentation of said antigen recognizing construct.

Item 32: The method according to item 30 or 31, wherein the geneticconstruct is an expression construct comprising a promoter sequenceoperably linked to said coding sequence.

Item 33: The method according to any one of items 30 to 32, wherein saidantigen recognizing construct is of mammalian origin, preferably ofhuman origin.

Item 34: The method according to any one of items 30 to 33, wherein saidsuitable host cell is a mammalian cell, optionally selected from a humancell or a human T lymphocyte.

Item 35: The method according to any of items 30 to 34, wherein saidantigen recognizing construct is a modified TCR, wherein saidmodification comprises addition of a functional domain comprising alabel, or an alternative domain comprising a membrane anchor domain.

Item 36: The method according to item 35, wherein said antigenrecognizing construct is an alpha/beta TCR, gamma/delta TCR, or a singlechain TCR (scTCR).

Item 37: The method according to any of items 30 to 36, wherein saidgenetic construct is introduced into said suitable host cell byretroviral transfection.

Item 38: The method according to any of items 30 to 37, furthercomprising the isolation and purification of the antigen recognizingconstruct from the suitable host cell and, optionally, reconstitution ofthe antigen recognizing construct in a T-cell.

The present invention will now be further described in the followingexamples with reference to the accompanying figures and sequences,nevertheless, without being limited thereto. For the purposes of thepresent invention, all references as cited herein are incorporated byreference in their entireties. In the Figures and Sequences:

FIG. 1: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R39P1C12 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) or variousSPINK2-001 alanine- or threonine-substitution variants at positions 1-9of SEQ ID NO:133 (SEQ ID NOs:134-149) or control peptide NYESO1-001 (SEQID NO:159). IFNγ release data were obtained with CD8+ T-cells derivedfrom two different healthy donors. RNA electroporated CD8+ T-cells aloneor in co-incubation with unloaded target cells served as controls.

FIG. 2: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R39P1F5 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) or variousSPINK2-001 alanine- or threonine-substitution variants at positions 1-9of SEQ ID NO:133 (SEQ ID NOs:134-149) or control peptide NYESO1-001 (SEQID NO:159). IFNγ release data were obtained with CD8+ T-cells derivedfrom two different healthy donors. RNA electroporated CD8+ T-cells aloneor in co-incubation with unloaded target cells served as controls.

FIG. 3: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R40P1C2 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) or variousSPINK2-001 alanine- or threonine-substitution variants at positions 1-9of SEQ ID NO:133 (SEQ ID NOs:134-149) or control peptide NYESO1-001 (SEQID NO:159). IFNγ release data were obtained with CD8+ T-cells derivedfrom two different healthy donors. RNA electroporated CD8+ T-cells aloneor in co-incubation with unloaded target cells served as controls.

FIG. 4: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R41P3E6 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) or variousSPINK2-001 alanine- or threonine-substitution variants at positions 1-9of SEQ ID NO:133 (SEQ ID NOs:134-149) or control peptide NYESO1-001 (SEQID NO:159). IFNγ release data were obtained with CD8+ T-cells derivedfrom two different healthy donors. RNA electroporated CD8+ T-cells aloneor in co-incubation with unloaded target cells served as controls.

FIG. 5: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R43P3G4 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) or variousSPINK2-001 alanine- or threonine-substitution variants at positions 1-9of SEQ ID NO:133 (SEQ ID NOs:134-149) or control peptide NYESO1-001 (SEQID NO:159). IFNγ release data were obtained with CD8+ T-cells derivedfrom two different healthy donors. RNA electroporated CD8+ T-cells aloneor in co-incubation with unloaded target cells served as controls.

FIG. 6: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R44P3B3 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) or variousSPINK2-001 alanine- or threonine-substitution variants at positions 1-9of SEQ ID NO:133 (SEQ ID NOs:134-149) or control peptide NYESO1-001 (SEQID NO:159). IFNγ release data were obtained with CD8+ T-cells derivedfrom two different healthy donors. RNA electroporated CD8+ T-cells aloneor in co-incubation with unloaded target cells served as controls.

FIG. 7: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R44P3E7 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) or variousSPINK2-001 alanine- or threonine-substitution variants at positions 1-9of SEQ ID NO:133 (SEQ ID NOs:134-149) or control peptide NYESO1-001 (SEQID NO:159). IFNγ release data were obtained with CD8+ T-cells derivedfrom two different healthy donors. RNA electroporated CD8+ T-cells aloneor in co-incubation with unloaded target cells served as controls.

FIG. 8: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R49P2B7 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) or variousSPINK2-001 alanine- or threonine-substitution variants at positions 1-9of SEQ ID NO:133 (SEQ ID NOs:134-149) or control peptide NYESO1-001 (SEQID NO:159). IFNγ release data were obtained with CD8+ T-cells derivedfrom two different healthy donors. RNA electroporated CD8+ T-cells aloneor in co-incubation with unloaded target cells served as controls.

FIG. 9: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R55P1G7 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) or variousSPINK2-001 alanine- or threonine-substitution variants at positions 1-9of SEQ ID NO:133 (SEQ ID NOs:134-149) or control peptide NYESO1-001 (SEQID NO:159). IFNγ release data were obtained with CD8+ T-cells derivedfrom two different healthy donors. RNA electroporated CD8+ T-cells aloneor in co-incubation with unloaded target cells served as controls.

FIG. 10: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R59P2A7 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) or variousSPINK2-001 alanine- or threonine-substitution variants at positions 1-9of SEQ ID NO:133 (SEQ ID NOs:134-149) or control peptide NYESO1-001 (SEQID NO:159). IFNγ release data were obtained with CD8+ T-cells derivedfrom two different healthy donors. RNA electroporated CD8+ T-cells aloneor in co-incubation with unloaded target cells served as controls.

FIG. 11: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCRs R39P1C12 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) orhomologous but unrelated peptide CYP-003 (SEQ ID NO:150), BAG6-001 (SEQID NO:151), XRCCS-001 (SEQ ID NO:152), TMEM147-001 (SEQ ID NO:153),SEC-001 (SEQ ID NO:154), GMPPA-001 (SEQ ID NO:155), EXOC4-002 (SEQ IDNO:156), ARFGAP3-001 (SEQ ID NO:157) or ANKRD29-002 (SEQ ID NO:158) orcontrol peptide NYESO1-001 (SEQ ID NO:159). IFNγ release data wereobtained with CD8+ T-cells derived from two different healthy donors.RNA electroporated CD8+ T-cells alone or in co-incubation with unloadedtarget cells served as controls.

FIG. 12: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCRs R39P1F5 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) orhomologous but unrelated peptide CYP-003 (SEQ ID NO:150), BAG6-001 (SEQID NO:151), XRCC5-001 (SEQ ID NO:152), TMEM147-001 (SEQ ID NO:153),SEC-001 (SEQ ID NO:154), GMPPA-001 (SEQ ID NO:155), EXOC4-002 (SEQ IDNO:156), ARFGAP3-001 (SEQ ID NO:157) or ANKRD29-002 (SEQ ID NO:158) orcontrol peptide NYESO1-001 (SEQ ID NO:159). IFNγ release data wereobtained with CD8+ T-cells derived from two different healthy donors.RNA electroporated CD8+ T-cells alone or in co-incubation with unloadedtarget cells served as controls.

FIG. 13: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCRs R40P1C2 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) orhomologous but unrelated peptide CYP-003 (SEQ ID NO:150), BAG6-001 (SEQID NO:151), XRCC5-001 (SEQ ID NO:152), TMEM147-001 (SEQ ID NO:153),SEC-001 (SEQ ID NO:154), GMPPA-001 (SEQ ID NO:155), EXOC4-002 (SEQ IDNO:156), ARFGAP3-001 (SEQ ID NO:157) or ANKRD29-002 (SEQ ID NO:158) orcontrol peptide NYESO1-001 (SEQ ID NO:159). IFNγ release data wereobtained with CD8+ T-cells derived from two different healthy donors.RNA electroporated CD8+ T-cells alone or in co-incubation with unloadedtarget cells served as controls.

FIG. 14: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCRs R41P3E6 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) orhomologous but unrelated peptide CYP-003 (SEQ ID NO:150), BAG6-001 (SEQID NO:151), XRCC5-001 (SEQ ID NO:152), TMEM147-001 (SEQ ID NO:153),SEC-001 (SEQ ID NO:154), GMPPA-001 (SEQ ID NO:155), EXOC4-002 (SEQ IDNO:156), ARFGAP3-001 (SEQ ID NO:157) or ANKRD29-002 (SEQ ID NO:158) orcontrol peptide NYESO1-001 (SEQ ID NO:159). IFNγ release data wereobtained with CD8+ T-cells derived from two different healthy donors.RNA electroporated CD8+ T-cells alone or in co-incubation with unloadedtarget cells served as controls.

FIG. 15: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCRs R43P3G4 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) orhomologous but unrelated peptide CYP-003 (SEQ ID NO:150), BAG6-001 (SEQID NO:151), XRCC5-001 (SEQ ID NO:152), TMEM147-001 (SEQ ID NO:153),SEC-001 (SEQ ID NO:154), GMPPA-001 (SEQ ID NO:155), EXOC4-002 (SEQ IDNO:156), ARFGAP3-001 (SEQ ID NO:157) or ANKRD29-002 (SEQ ID NO:158) orcontrol peptide NYESO1-001 (SEQ ID NO:159). IFNγ release data wereobtained with CD8+ T-cells derived from two different healthy donors.RNA electroporated CD8+ T-cells alone or in co-incubation with unloadedtarget cells served as controls.

FIG. 16: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCRs R44P3B3 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) orhomologous but unrelated peptide CYP-003 (SEQ ID NO:150), BAG6-001 (SEQID NO:151), XRCC5-001 (SEQ ID NO:152), TMEM147-001 (SEQ ID NO:153),SEC-001 (SEQ ID NO:154), GMPPA-001 (SEQ ID NO:155), EXOC4-002 (SEQ IDNO:156), ARFGAP3-001 (SEQ ID NO:157) or ANKRD29-002 (SEQ ID NO:158) orcontrol peptide NYESO1-001 (SEQ ID NO:159). IFNγ release data wereobtained with CD8+ T-cells derived from two different healthy donors.RNA electroporated CD8+ T-cells alone or in co-incubation with unloadedtarget cells served as controls.

FIG. 17: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCRs R44P3E7 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) orhomologous but unrelated peptide CYP-003 (SEQ ID NO:150), BAG6-001 (SEQID NO:151), XRCC5-001 (SEQ ID NO:152), TMEM147-001 (SEQ ID NO:153),SEC-001 (SEQ ID NO:154), GMPPA-001 (SEQ ID NO:155), EXOC4-002 (SEQ IDNO:156), ARFGAP3-001 (SEQ ID NO:157) or ANKRD29-002 (SEQ ID NO:158) orcontrol peptide NYESO1-001 (SEQ ID NO:159). IFNγ release data wereobtained with CD8+ T-cells derived from two different healthy donors.RNA electroporated CD8+ T-cells alone or in co-incubation with unloadedtarget cells served as controls.

FIG. 18: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCRs R49P2B7 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) orhomologous but unrelated peptide CYP-003 (SEQ ID NO:150), BAG6-001 (SEQID NO:151), XRCC5-001 (SEQ ID NO:152), TMEM147-001 (SEQ ID NO:153),SEC-001 (SEQ ID NO:154), GMPPA-001 (SEQ ID NO:155), EXOC4-002 (SEQ IDNO:156), ARFGAP3-001 (SEQ ID NO:157) or ANKRD29-002 (SEQ ID NO:158) orcontrol peptide NYESO1-001 (SEQ ID NO:159). IFNγ release data wereobtained with CD8+ T-cells derived from two different healthy donors.RNA electroporated CD8+ T-cells alone or in co-incubation with unloadedtarget cells served as controls.

FIG. 19: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCRs R55P1G7 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) orhomologous but unrelated peptide CYP-003 (SEQ ID NO:150), BAG6-001 (SEQID NO:151), XRCC5-001 (SEQ ID NO:152), TMEM147-001 (SEQ ID NO:153),SEC-001 (SEQ ID NO:154), GMPPA-001 (SEQ ID NO:155), EXOC4-002 (SEQ IDNO:156), ARFGAP3-001 (SEQ ID NO:157) or ANKRD29-002 (SEQ ID NO:158) orcontrol peptide NYESO1-001 (SEQ ID NO:159). IFNγ release data wereobtained with CD8+ T-cells derived from two different healthy donors.RNA electroporated CD8+ T-cells alone or in co-incubation with unloadedtarget cells served as controls.

FIG. 20: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCRs R59P2A7 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) orhomologous but unrelated peptide CYP-003 (SEQ ID NO:150), BAG6-001 (SEQID NO:151), XRCC5-001 (SEQ ID NO:152), TMEM147-001 (SEQ ID NO:153),SEC-001 (SEQ ID NO:154), GMPPA-001 (SEQ ID NO:155), EXOC4-002 (SEQ IDNO:156), ARFGAP3-001 (SEQ ID NO:157) or ANKRD29-002 (SEQ ID NO:158) orcontrol peptide NYESO1-001 (SEQ ID NO:159). IFNγ release data wereobtained with CD8+ T-cells derived from two different healthy donors.RNA electroporated CD8+ T-cells alone or in co-incubation with unloadedtarget cells served as controls.

FIG. 21: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R39P1C12 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 μM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 22: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R39P1F5 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 μM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 23: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R40P1C2 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 μM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 24: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R41P3E6 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 μM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 25: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R43P3G4 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 μM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 26: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R44P3B3 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 μM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 27: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R44P3E7 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 μM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 28: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R49P2B7 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 μM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 29: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R55P1G7 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 μM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 30: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R59P2A7 (Table 1) after co-incubation with T2target cells loaded with SPINK2-001 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 μM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 31: HLA-A*02/SPINK2-001 tetramer or HLA-A*02/NYESO1-001 tetramerstaining, respectively, of CD8+ T-cells electroporated with alpha andbeta chain RNA of TCRs R41P3E6, R44P3B3, R49P2B7 and R55P1G7,respectively. CD8+ T-cells electroporated with RNA of 1G4 TCR (SEQ ID:121-132) that specifically binds to HLA-A*02/NYESO1-001 complex and mockelectroporated CD8+ T-cells served as controls.

FIG. 32: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R55P1G7 (A) or R44P3B3 (B) (Table 1) afterco-incubation with A375 target cells overexpressing SPINK2 or wildtypeA375 cells. IFNγ release data were obtained with CD8+ T-cells derivedfrom two different healthy donors (TCRA-61 and TCRA-62). Backgroundsubtracted data is shown (i.e. pre-activation from effector onlycontrols and signal of non-transduced effector co-cultured with targetcell line was subtracted from samples of interest).

FIG. 33: Biolayer interferometry binding analysis ofHLA-A*02:01:SPINK2-001 (immobilized on sensor) and soluble TCR variantsof TCRs R39P1F5, R39P1C12, R41P3E6, R44P3B3 and R55P1G7. Left panelshows the signal (y-axis) of binding curves and fits for different TCRconcentrations over time (x-axis), right panel shows the response at theequilibrium (y-axis) over different TCR concentrations (x-axis) andfits, respectively.

TABLE 1 TCR sequences of the invention SEQ ID NO: TCR Chain RegionSequence 1 R39P1C12 alpha CDR1 DSSSTY 2 R39P1C12 alpha CDR2 IFS 3R39P1C12 alpha CDR3 CAEIDNQGGKLIF 4 R39P1C12 alpha variable domainMKTFAGFSFLFLWLQLDCMSRGED VEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMD MKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEIDNQGGKLIFGQGT ELSVKP 5 R39P1C12 alpha constant domainNIQNPDPAVYQLRDSKSSDKSVCLFT DFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 6 R39P1C12 alpha full-lengthMKTFAGFSFLFLWLQLDCMSRGED VEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMD MKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEIDNQGGKLIFGQGT ELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCD VKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 7 R39P1C12 beta CDR1 SGHDT 8 R39P1C12 beta CDR2YYEEEE 9 R39P1C12 beta CDR3 CASSQLNTEAFF 10 R39P1C12 betavariable domain MGPGLLCWALLCLLGAGLVDAGVT QSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQ RGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSQLNTEAFFGQGT RLTVV 11 R39P1C12 beta constant domainEDLNKVFPPEVAVFEPSEAEISHTQK ATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKR KDF 12 R39P1C12 beta full-lengthMGPGLLCWALLCLLGAGLVDAGVT QSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQ RGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSQLNTEAFFGQGT RLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSW WVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHF RCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVL SATILYEILLGKATLYAVLVSALVLM AMVKRKDF 13 R39P1F5alpha CDR1 DRGSQS 14 R39P1F5 alpha CDR2 IY 15 R39P1F5 alpha CDR3CAVNNARLMF 16 R39P1F5 alpha variable domain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRG SQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ PSDSATYLCAVNNARLMFGDGTQL VVKP 17 R39P1F5 alphaconstant domain NIQNPDPAVYQLRDSKSSDKSVCLFT DFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 18 R39P1F5 alpha full-lengthMKSLRVLLVILWLQLSWVWSQQKE VEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGD KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNNARLMFGDGTQL VVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITD KTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDV KLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 19 R39P1F5 beta CDR1 SNHLY 20 R39P1F5 beta CDR2FYNNEI 21 R39P1F5 beta CDR3 CASSGQGANEQYF 22 R39P1F5 betavariable domain MDTWLVCWAIFSLLKAGLTEPEVTQ TPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKS EIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSGQGANEQYFGPGTRL TVT 23 R39P1F5 beta constant domainEDLKNVFPPEVAVFEPSEAEISHTQK ATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG 24 R39P1F5 beta full-lengthMDTWLVCWAIFSLLKAGLTEPEVTQ TPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKS EIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSGQGANEQYFGPGTRL TVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV NGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQ VQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATI LYEILLGKATLYAVLVSALVLMAM VKRKDSRG 25 R40P1C2alpha CDR1 TSESDYY 26 R40P1C2 alpha CDR2 QEAY 27 R40P1C2 alpha CDR3CAYLNYQLIW 28 R40P1C2 alpha variable domain MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSES DYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKIS DSQLGDAAMYFCAYLNYQLIWGAG TKLIIKP 29 R40P1C2alpha constant domain DIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLD MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEK SFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 30R40P1C2 alpha full-length MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSES DYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKIS DSQLGDAAMYFCAYLNYQLIWGAGTKLIIKPDIQNPDPAVYQLRDSKSSD KSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKS DFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRI LLLKVAGFNLLMTLRLWSS 31 R40P1C2 beta CDR1SNHLY 32 R40P1C2 beta CDR2 FYNNEI 33 R40P1C2 beta CDR3 CASSEMTAVGQYF 34R40P1C2 beta variable domain MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYF YWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLE DSAMYFCASSEMTAVGQYFGPGTR LTVT 35 R40P1C2beta constant domain EDLKNVFPPEVAVFEPSEAEISHTQK ATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG 36 R40P1C2 beta full-lengthMDTWLVCWAIFSLLKAGLTEPEVTQ TPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKS EIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSEMTAVGQYFGPGTR LTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWW VNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRC QVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSAT ILYEILLGKATLYAVLVSALVLMAM VKRKDSRG 37 R41P3E6alpha CDR1 DRGSQS 38 R41P3E6 alpha CDR2 IY 39 R41P3E6 alpha CDR3CAAFSGYALNF 40 R41P3E6 alpha variable domain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRG QSSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ PSDSATYLCAAFSGYALNFGKGTSL LVTP 41 R41P3E6alpha constant domain HIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLD MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEK SFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 42R41P3E6 alpha full-length MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRG SQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ PSDSATYLCAAFSGYALNFGKGTSLLVTPHIQNPDPAVYQLRDSKSSDKS VCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSD FACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL LLKVAGFNLLMTLRLWSS 43 R41P3E6 beta CDR1 SNHLY44 R41P3E6 beta CDR2 FYNNEI 45 R41P3E6 beta CDR3 CASSQYTGELFF 46 R41P3E6beta variable domain MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYF WYYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLE DSAMYFCASSQYTGELFFGEGSRLT VL 47 R41P3E6 betaconstant domain EDLKNVFPPEVAVFEPSEAEISHTQK ATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG 48 R41P3E6 beta full-lengthMDTWLVCWAIFSLLKAGLTEPEVTQ TPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKS EIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSQYTGELFFGEGSRLT VLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVN GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQV QFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATIL YEILLGKATLYAVLVSALVLMAMV KRKDSRG 49 R43P3G4 alpha CDR1 DRGSQS 50 R43P3G4 alpha CDR2 IY 51 R43P3G4 alpha CDR3CAVNGGDMRF 52 R43P3G4 alpha variable domain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRG SQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ PSDSATYLCAVNGGDMRFGAGTRL TVKP 53 R43P3G4 alphaconstant domain NIQNPDPAVYQLRDSKSSDKSVCLFT DFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 54 R43P3G4 alpha full-lengthMKSLRVLLVILWLQLSWVWSQQKE VEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGD KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNGGDMRFGAGTRL TVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITD KTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDV KLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 55 R43P3G4 beta CDR1 SNHLY 56 R43P3G4 beta CDR2FYNNEI 57 R43P3G4 beta CDR3 CASSGQGALEQYF 58 R43P3G4 betavariable domain MDTWLVCWAIFSLLKAGLTEPEVTQ TPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKS EIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSGQGALEQYFGPGTRL TVT 59 R43P3G4 beta constant domainEDLKNVFPPEVAVFEPSEAEISHTQK ATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG 60 R43P3G4 beta full-lengthMDTWLVCWAIFSLLKAGLTEPEVTQ TPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKS EIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSGQGALEQYFGPGTRL TVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV NGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQ VQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATI LYEILLGKATLYAVLVSALVLMAM VKRKDSRG 61 R44P3B3alpha CDR1 NSMFDY 62 R44P3B3 alpha CDR2 ISS 63 R44P3B3 alpha CDR3CAASGLYNQGGKLIF 64 R44P3B3 alpha variable domainMAMLLGASVLILWLQPDWVNSQQK NDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISI SSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGLYNQGGK LIFGQGTELSVKP 65 R44P3B3 alpha constant domainNIQNPDPAVYQLRDSKSSDKSVCLFT DFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 66 R44P3B3 alpha full-lengthMAMLLGASVLILWLQPDWVNSQQK NDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISI SSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGLYNQGGK LIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSK DSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFP SPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 67 R44P3B3 beta CDR1 LGHDT 68 R44P3B3 betaCDR2 YNNKEL 69 R44P3B3 beta CDR3 CASSLGDRGYEQYF 70 R44P3B3 betavariable domain MGCRLLCCVVFCLLQAGPLDTAVSQ TPKYLVTQMGNDKSIKCEQNLGHDTMYWYKQDSKKFLKIMFSYNNKELII NETVPNRFSPKSPDKAHLNLHINSLELGDSAVYFCASSLGDRGYEQYFGPG TRLTVT 71 R44P3B3 beta constant domainEDLKNVFPPEVAVFEPSEAEISHTQK ATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG 72 R44P3B3 beta full-lengthMGCRLLCCVVFCLLQAGPLDTAVSQ TPKYLVTQMGNDKSIKCEQNLGHDTMYWYKQDSKKFLKIMFSYNNKELII NETVPNRFSPKSPDKAHLNLHINSLELGDSAVYFCASSLGDRGYEQYFGPG TRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSW WVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHF RCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVL SATILYEILLGKATLYAVLVSALVLM AMVKRKDSRG 73R44P3E7 alpha CDR1 DSSSTY 74 R44P3E7 alpha CDR2 IFS 75 R44P3E7 alphaCDR3 CAEINNNARLMF 76 R44P3E7 alpha variable domainMKTFAGFSFLFLWLQLDCMSRGED VEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMD MKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEINNNARLMFGDGT QLVVKP 77 R44P3E7 alpha constant domainNIQNPDPAVYQLRDSKSSDKSVCLFT DFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 78 R44P3E7 alpha full-lengthMKTFAGFSFLFLWLQLDCMSRGED VEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMD MKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEINNNARLMFGDGT QLVVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCD VKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 79 R44P3E7 beta CDR1 PRHDT 80 R44P3E7 beta CDR2FYEKMQ 81 R44P3E7 beta CDR3 CASSPPDQNTQYF 82 R44P3E7 betavariable domain MLSPDLPDSAWNTRLLCHVMLCLL GAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFL ISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSPPDQ NTQYFGPGTRLTVL 83 R44P3E7 betaconstant domain EDLKNVFPPEVAVFEPSEAEISHTQK ATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG 84 R44P3E7 beta full-lengthMLSPDLPDSAWNTRLLCHVMLCLL GAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFL ISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSPPDQ NTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFY PDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATF WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS ESYQQGVLSATILYEILLGKATLYAV LVSALVLMAMVKRKDSRG85 R49P2B7 alpha CDR1 SSVPPY 86 R49P2B7 alpha CDR2 YTTG 87 R49P2B7 alphaCDR3 CAVRIFGNEKLTF 88 R49P2B7 alpha variable domainMLLLLVPVLEVIFTLGGTRAQSVTQL GSHVSVSEGALVLLRCNYSSSVPPYLFWYVQYPNQGLQLLLKYTTGATLV KGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCAVRIFGNEKLTFGTGTRL TIIP 89 R49P2B7 alpha constant domainNIQNPDPAVYQLRDSKSSDKSVCLFT DFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 90 R49P2B7 alpha full-lengthMLLLLVPVLEVIFTLGGTRAQSVTQL GSHVSVSEGALVLLRCNYSSSVPPYLFWYVQYPNQGLQLLLKYTTGATLV KGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCAVRIFGNEKLTFGTGTRL TIIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKL VEKSFETDTNLNFQNLSVIGFRILLL KVAGFNLLMTLRLWSS91 R49P2B7 beta CDR1 MDHEN 92 R49P2B7 beta CDR2 SYDVKM 93 R49P2B7 betaCDR3 CASSLMGELTGELFF 94 R49P2B7 beta variable domainMGIRLLCRVAFCFLAVGLVDVKVTQ SSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKM KEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLMGELTGELFF GEGSRLTVL 95 R49P2B7 beta constant domainEDLKNVFPPEVAVFEPSEAEISHTQK ATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG 96 R49P2B7 beta full-lengthMGIRLLCRVAFCFLAVGLVDVKVTQ SSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKM KEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLMGELTGELFF GEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVE LSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPR NHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQ GVLSATILYEILLGKATLYAVLVSAL VLMAMVKRKDSRG 97R55P1G7 alpha CDR1 NSAFQY 98 R55P1G7 alpha CDR2 TY 99 R55P1G7 alpha CDR3CAMMGDTGTASKLTF 100 R55P1G7 alpha variable domainMMKSLRVLLVILWLQLSWVWSQQK EVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYS SGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMMGDTGTASKLTF GTGTRLQVTL 101 R55P1G7 alpha constant domainDIQNPDPAVYQLRDSKSSDKSVCLFT DFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 102 R55P1G7 alpha full-lengthMMKSLRVLLVILWLQLSWVWSQQK EVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYS SGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMMGDTGTASKLTF GTGTRLQVTLDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDS DVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPS PESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 103 R55P1G7 beta CDR1 MDHEN 104 R55P1G7 betaCDR2 SYDVKM 105 R55P1G7 beta CDR3 CASSFGGYEQYF 106 R55P1G7 betavariable domain MGIRLLCRVAFCFLAVGLVDVKVTQ SSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKM KEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSFGGYEQYFGPG TRLTVT 107 R55P1G7 beta constant domainEDLKNVFPPEVAVFEPSEAEISHTQK ATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG 108 R55P1G7 beta full-lengthMGIRLLCRVAFCFLAVGLVDVKVTQ SSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKM KEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSFGGYEQYFGPG TRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSW WVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHF RCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVL SATILYEILLGKATLYAVLVSALVLM AMVKRKDSRG 109R59P2A7 alpha CDR1 DRGSQS 110 R59P2A7 alpha CDR2 IY 111 R59P2A7 alphaCDR3 CAVQPHDMRF 112 R59P2A7 alpha variable domainMKSLRVLLVILWLQLSWVWSQQKE VEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGD KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVQPHDMRFGAGTRLT VKP 113 R59P2A7 alpha constant domainNIQNPDPAVYQLRDSKSSDKSVCLFT DFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 114 R59P2A7 alpha full-lengthMKSLRVLLVILWLQLSWVWSQQKE VEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGD KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVQPHDMRFGAGTRLT VKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKL VEKSFETDTNLNFQNLSVIGFRILLL KVAGFNLLMTLRLWSS115 R59P2A7 beta CDR1 GTSNPN 116 R59P2A7 beta CDR2 SVGIG 117 R59P2A7beta CDR3 CAWSGLVAEQFF 118 R59P2A7 beta variable domainMLCSLLALLLGTFFGVRSQTIHQWP ATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSE VPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWSGLVAEQFFGPGTRLTV L 119 R59P2A7 beta constant domainEDLKNVFPPEVAVFEPSEAEISHTQK ATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG 120 R59P2A7 beta full-lengthMLCSLLALLLGTFFGVRSQTIHQWP ATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSE VPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWSGLVAEQFFGPGTRLTV LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNG KEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQ FYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILY EILLGKATLYAVLVSALVLMAMVK RKDSRG 121 1G4 alphaCDR1 DSAIYN 122 1G4 alpha CDR2 IQS 123 1G4 alpha CDR3 CAVRPTSGGSYIPTF124 1G4 alpha variable domain METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYN LQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGD SATYLCAVRPTSGGSYIPTFGRGTSLI VHP 125 1G4 alphaconstant domain YIQNPDPAVYQLRDSKSSDKSVCLFT DFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 126 1G4 alpha full-lengthMETLLGLLILWLQLQWVSSKQEVTQ IPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQT SGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLI VHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKL VEKSFETDTNLNFQNLSVIGFRILLL KVAGFNLLMTLRLWSS127 1G4 beta CDR1 MNHEY 128 1G4 beta CDR2 SVGAGI 129 1G4 beta CDR3CASSYVGNTGELFF 130 1G4 beta variable domain MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHE YMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLS AAPSQTSVYFCASSYVGNTGELFFG EGSRLTVL 131 1G4beta constant domain EDLKNVFPPEVAVFEPSEAEISHTQK ATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DSRG 132 1G4 beta full-lengthMSIGLLCCAALSLLWAGPVNAGVTQ TPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGI TDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFG EGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELS WWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNH FRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGV LSATILYEILLGKATLYAVLVSALVL MAMVKRKDSRG

TABLE 2 Peptide sequences of the invention Peptide Code SequenceSEQ ID NO: SPINK2-001 ALSVLRLAL 133 SPINK2-001_A2 AASVLRLAL 134SPINK2-001_A3 ALAVLRLAL 135 SPINK2-001_A4 ALSALRLAL 136 SPINK2-001_A5ALSVARLAL 137 SPINK2-001_A6 ALSVLALAL 138 SPINK2-001_A7 ALSVLRAAL 139SPINK2-001_A9 ALSVLRLAA 140 SPINK2-001_T1 TLSVLRLAL 141 SPINK2-001_T2ATSVLRLAL 142 SPINK2-001_T3 ALTVLRLAL 143 SPINK2-001_T4 ALSTLRLAL 144SPINK2-001_T5 ALSVTRLAL 145 SPINK2-001_T6 ALSVLTLAL 146 SPINK2-001_T7ALSVLRTAL 147 SPINK2-001_T8 ALSVLRLTL 148 SPINK2-001_T9 ALSVLRLAT 149CYP-003 ALMNMKLAL 150 BAG6-001 ALSDLRCNL 151 XRCC5-001 ALSSLIHAL 152TMEM147-001 ALSTLALYV 153 SEC-001 ALSVLADFL 154 GMPPA-001 ALYASRLYL 155EXOC4-002 GLSDLRLEL 156 ARFGAP3-001 IVSSLRLAY 157 ANKRD29-002 YLDVIRLLL158 NYESO1-001 SLLMWITQV 159

EXAMPLES

Ten SPINK2-001-specific TCRs (R39P1C12, R39P1F5, R40P1C2, R41P3E6,R43P3G4, R44P3B3, R44P3E7, R49P2B7, R55P1G7 and R59P2A7, see Table 1),each encoding tumor specific TCR-alpha and TCR-beta chains, wereisolated and amplified from T-cells of healthy donors. Cells fromhealthy donors were in vitro stimulated according to a method previouslydescribed (Walter et al., 2003 J Immunol., November 15; 171(10):4974-8)and target-specific cells were single-cell sorted using HLA-A*02multimers and then used for subsequent TCR isolation. TCR sequences wereisolated via 5′ RACE by standard methods as described by e.g. MolecularCloning a laboratory manual fourth edition by Green and Sambrook. Thealpha and beta variable regions of TCRs R39P1C12, R39P1F5, R40P1C2,R41P3E6, R43P3G4, R44P3B3, R44P3E7, R49P2B7, R55P1G7 and R59P2A7 weresequenced and expression constructs were generated by gene synthesis forfurther functional characterization.

R41P3E6, R43P3G4, R55P1G7 and R59P2A7 are derived from HLA-A*02 negativedonor (allo-reactive setting) and R39P1C12, R39P1F5, R40P1C2, R44P3B3,R44P3E7 and R49P2B7 are derived from a HLA-A*02 positive donor.

TCRs of interest were expressed in human T cells, e.g. through mRNAelectroporation. The T cells were assessed for IFN-γ release afterco-culture with different target cells, such as T2 cells loaded withdifferent peptides as well as tumor cell lines. For T-cell activation,data is either shown as absolute IFNγ levels or as background subtracteddata, indicated below. Efficacy of CD8+ T cells expressing TCRs R55P1G7and R44P3B3 was determined e.g. by T cell activation studies (IFNγrelease) using different tumor cell lines as target cells.

Background subtraction method for IFNγ release:

Meanbg(TCRoi;co)=[mean(TCRoi;co)−mean(TCRoi;effectoronly)]−[mean(mock;co)mean(mock;effector only)]

The respective SDbg was calculated:

SDbg(TCRoi;co)=[SD(TCRoi;co)²+SD(TCRoi;effectoronly)²+SD(mock;co)²+SD(mock;effector only)²]{circumflex over( )}^([1/2])

TCRoi=effector cells expressing TCR of interestMock=effector cells without exogenous TCR expressionCo=effector cells co-cultured with target cellsEffector only=effector cells not co-culturedMean(bg)=mean IFNγ release (background subtracted)SD(bg)=standard deviation (background subtracted)

BLI (Biolayer interferometry) binding analysis for SPINK2-001 TCRs,expressed as soluble TCRs according to a previously described method(Willcox B E et al., 1999 Protein Sci., November; 8(11):2418-23), andHLA-A*02/SPINK2-001 complex was used for affinity determination. BLIbinding data for 1G4 TCR and HLA-A*02/NYESO1-001 are used as control.

Example 1: T-Cell Receptor R39P1C12

TCR R39P1C12 (SEQ ID NOs: 1-12) is restricted towards HLA-A*02-presentedSPINK2-001 (SEQ ID NO: 133) (see FIG. 11).

R39P1C12 specifically recognizes SPINK2-001 as human primary CD8+T-cells re-expressing this TCR release IFNγ upon co-incubation withHLA-A*02+ target cells, loaded either with SPINK2-001 peptide or alanineor threonine substitution variants of SPINK2-001 (FIG. 1) or differentpeptides showing high degree of sequence similarity to SPINK2-001 (FIG.11). NYESO1-001 peptide is used as negative control. TCR R39P1C12 has anEC50 of 0.81 nM (FIG. 21) and an affinity of 18 μM (R²=0.9956) (FIG.33).

Example 2: T-Cell Receptor R39P1F5

TCR R39P1F5 (SEQ ID NOs: 13-24) is restricted towards HLA-A*02-presentedSPINK2-001 (SEQ ID NO: 133) (see FIG. 12).

R39P1F5 specifically recognizes SPINK2-001 as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells, loaded either with SPINK2-001 peptide or alanine orthreonine substitution variants of SPINK2-001 (FIG. 2) or differentpeptides showing high degree of sequence similarity to SPINK2-001 (FIG.12). NYESO1-001 peptide is used as negative control. TCR R39P1F5 has anEC₅₀ of 1.52 nM (FIG. 22) and an affinity of 34 μM (R²=0.9962) (FIG.33).

Example 3: T-Cell Receptor R40P1C2

TCR R40P1C2 (SEQ ID NOs: 25-36) is restricted towards HLA-A*02-presentedSPINK2-001 (SEQ ID NO: 133) (see FIG. 13).

R40P1C2 specifically recognizes SPINK2-001 as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells, loaded either with SPINK2-001 peptide or alanine orthreonine substitution variants of SPINK2-001 (FIG. 3) or differentpeptides showing high degree of sequence similarity to SPINK2-001 (FIG.13). NYESO1-001 peptide is used as negative control. TCR R40P1C2 has anEC₅₀ of 1.94 nM (FIG. 23).

Example 4: T-Cell Receptor R41P3E6

TCR R41P3E6 (SEQ ID NOs: 37-48) is restricted towards HLA-A*02-presentedSPINK2-001 (SEQ ID NO: 133) (see FIG. 14).

R41P3E6 specifically recognizes SPINK2-001 as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells and bind HLA-A*02 tetramers (FIG. 31), respectively, loadedeither with SPINK2-001 peptide or alanine or threonine substitutionvariants of SPINK2-001 (FIG. 4) or different peptides showing highdegree of sequence similarity to SPINK2-001 (FIG. 14). NYESO1-001peptide is used as negative control. TCR R41P3E6 has an EC₅₀ of 1.03 nM(FIG. 24) and an affinity of 13 μM (R²=0.9892) (FIG. 33).

Example 5: T-Cell Receptor R43P3G4

TCR R43P3G4 (SEQ ID NOs: 49-60) is restricted towards HLA-A*02-presentedSPINK2-001 (SEQ ID NO: 133) (see FIG. 15).

R43P3G4 specifically recognizes SPINK2-001 as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells, loaded either with SPINK2-001 peptide or alanine orthreonine substitution variants of SPINK2-001 (FIG. 5) or differentpeptides showing high degree of sequence similarity to SPINK2-001 (FIG.15). NYESO1-001 peptide is used as negative control. TCR R43P3G4 has anEC₅₀ of 1.34 nM (FIG. 25).

Example 6: T-Cell Receptor R44P3B3

TCR R44P3B3 (SEQ ID NOs: 61-72) is restricted towards HLA-A*02-presentedSPINK2-001 (SEQ ID NO: 133) (see FIG. 16).

R44P3B3 specifically recognizes SPINK2-001 as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells and bind HLA-A*02 tetramers (FIG. 31), respectively, loadedeither with SPINK2-001 peptide or alanine or threonine substitutionvariants of SPINK2-001 (FIG. 6) or different peptides showing highdegree of sequence similarity to SPINK2-001 (FIG. 16). NYESO1-001peptide is used as negative control. TCR R44P3B3 has an EC₅₀ of 1.06 nM(FIG. 26) and an affinity of 37 μM (R²=0.9947) (FIG. 33).

For CD8+ T cells expressing TCR R44P3B3, an activity towards theSPINK2-001-overexpressing A375 tumor cells was observed (FIG. 32), butnot towards the wildtype A375 tumor cell line. A375 cells endogenouslyexpress HLA-A2.

T-cell activation upon co-culture with cell lines expressing HLA-A*02and SPINK2-001 reflects the recognition of endogenously presented targetpHLA (peptide presented on human leukocyte antigen) by TCR R44P3B3.

Example 7: T-Cell Receptor R44P3E7

TCR R44P3E7 (SEQ ID NOs: 73-84) is restricted towards HLA-A*02-presentedSPINK2-001 (SEQ ID NO: 133) (see FIG. 17).

R44P3E7 specifically recognizes SPINK2-001 as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells, loaded either with SPINK2-001 peptide or alanine orthreonine substitution variants of SPINK2-001 (FIG. 7) or differentpeptides showing high degree of sequence similarity to SPINK2-001 (FIG.17). NYESO1-001 peptide is used as negative control. TCR R44P3E7 has anEC₅₀ of 0.86 nM (FIG. 27).

Example 8: T-Cell Receptor R49P2A7

TCR R49P2A7 (SEQ ID NOs: 85-96) is restricted towards HLA-A*02-presentedSPINK2-001 (SEQ ID NO: 133) (see FIG. 18).

R49P2A7 specifically recognizes SPINK2-001 as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells and bind HLA-A*02 tetramers (FIG. 31), respectively, loadedeither with SPINK2-001 peptide or alanine or threonine substitutionvariants of SPINK2-001 (FIG. 8) or different peptides showing highdegree of sequence similarity to SPINK2-001 (FIG. 18). NYESO1-001peptide is used as negative control. TCR R49P2A7 has an EC₅₀ of >83.24nM (FIG. 28).

Example 9: T-Cell Receptor R55P1G7

TCR R55P1G7 (SEQ ID NOs: 97-108) is restricted towardsHLA-A*02-presented SPINK2-001 (SEQ ID NO: 133) (see FIG. 19).

R55P1G7 specifically recognizes SPINK2-001 as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells and bind HLA-A*02 tetramers (FIG. 31), respectively, loadedeither with SPINK2-001 peptide or alanine or threonine substitutionvariants of SPINK2-001 (FIG. 9) or different peptides showing highdegree of sequence similarity to SPINK2-001 (FIG. 19). NYESO1-001peptide is used as negative control. TCR R55P1G7 has an EC₅₀ of 91.5 μM(FIG. 29) and an affinity of 4.3 μM (R²=0.9765) (FIG. 33).

For CD8+ T cells expressing TCR R55P1G7, an activity towards theSPINK2-001-overexpressing A375 tumor cells was observed (FIG. 32), butnot towards the wildtype A375 tumor cell line. The A375 cellsendogenously express HLA-A2.

T-cell activation upon co-culture with cell lines expressing HLA-A*02and SPINK2-001 reflects the recognition of endogenously presented targetpHLA (peptide presented on human leukocyte antigen) by TCR R55P1G7.

Example 10: T-Cell Receptor R59P2A7

TCR R59P2A7 (SEQ ID NOs: 109-120) is restricted towardsHLA-A*02-presented SPINK2-001 (SEQ ID NO: 133) (see FIG. 20).

R59P2A7 specifically recognizes SPINK2-001 as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells, loaded either with SPINK2-001 peptide or alanine orthreonine substitution variants of SPINK2-001 (FIG. 10) or differentpeptides showing high degree of sequence similarity to SPINK2-001 (FIG.20). NYESO1-001 peptide is used as negative control. TCR R59P2A7 has anEC₅₀ of 0.86 nM (FIG. 30).

1. A method of treating a patient who has cancer, comprisingadministering to the patient a population of transformed T cellsexpressing at least one vector encoding a T cell receptor (TCR), whereinthe TCR comprises SEQ ID NOs: 1, 3, 7, and 9, or SEQ ID NOs: 13, 15, 19,and 21, or SEQ ID NOs: 25, 27, 31, and 33, or SEQ ID NOs: 37, 39, 43,and 45, or SEQ ID NOs: 49, 51, 55, and 57, or SEQ ID NOs: 61, 63, 67,and 69, or SEQ ID NOs: 73, 75, 79, and 81, or SEQ ID NOs: 85, 87, 91,and 93, or SEQ ID NOs: 97, 99, 103, and 105, or SEQ ID NOs: 109, 111,115, and 117, wherein each of SEQ ID NOs: 1, 3, 7, 9, 13, 15, 19, 21,25, 27, 31, 33, 37, 39, 43, 45, 49, 51, 55, 57, 61, 63, 67, 69, 73, 75,79, 81, 85, 87, 91, 93, 97, 99, 103, 105, 109, 111, 115, and 117comprises at most one conservative amino acid substitution, wherein theTCR binds to a peptide consisting of the amino acid sequence ofALSVLRLAL (SEQ ID NO: 133) in a complex with an MHC class I molecule,and wherein the cancer is selected from acute lymphocytic cancer, acutemyeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer,breast cancer, cancer of the anus, anal canal, or anorectum, cancer ofthe eye, cancer of the intrahepatic bile duct, cancer of the joints,cancer of the neck, gallbladder, or pleura, cancer of the nose, nasalcavity, or middle ear, cancer of the oral cavity, cancer of the vagina,cancer of the vulva, chronic lymphocytic leukemia, chronic myeloidcancer, colon cancer, esophageal cancer, cervical cancer,gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharynxcancer, kidney cancer, larynx cancer, liver cancer, lung cancer,malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer,non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancerof the penis, pancreatic cancer, peritoneum, omentum, and mesenterycancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer,skin cancer, small intestine cancer, soft tissue cancer, stomach cancer,testicular cancer, thyroid cancer, cancer of the uterus, ureter cancer,and urinary bladder cancer.
 2. The method of claim 1, wherein thepopulation of transformed cells are produced by a method comprisingisolating a CD8+ T cell from a subject, transforming the cell with atleast one vector encoding the TCR to produce a transformed cell, andexpanding the transformed cell to produce the population of transformedcells.
 3. The method of claim 2, wherein the subject is the patient. 4.The method of claim 1, wherein said T cells are CD8+ T cells.
 5. Themethod of claim 1, wherein the TCR comprises an α chain comprising theamino acid sequence of SEQ ID NO: 6 and a β chain comprising the aminoacid sequence of SEQ ID NO: 12, or an α chain comprising the amino acidsequence of SEQ ID NO: 18 and a β chain comprising the amino acidsequence of SEQ ID NO: 24, or an α chain comprising the amino acidsequence of SEQ ID NO: 30 and a β chain comprising of the amino acidsequence of SEQ ID NO: 36, or an α chain comprising the amino acidsequence of SEQ ID NO: 42 and a β chain comprising of the amino acidsequence of SEQ ID NO: 48, or an α chain comprising the amino acidsequence of SEQ ID NO: 54 and a β chain comprising of the amino acidsequence of SEQ ID NO: 60, or an α chain comprising the amino acidsequence of SEQ ID NO: 66 and a β chain comprising of the amino acidsequence of SEQ ID NO: 72, or an α chain comprising the amino acidsequence of SEQ ID NO: 78 and a β chain comprising of the amino acidsequence of SEQ ID NO: 84, or an α chain comprising the amino acidsequence of SEQ ID NO: 90 and a β chain comprising of the amino acidsequence of SEQ ID NO: 96, or an α chain comprising the amino acidsequence of SEQ ID NO: 102 and a β chain comprising of the amino acidsequence of SEQ ID NO: 108, or an α chain comprising the amino acidsequence of SEQ ID NO: 114 and a β chain comprising of the amino acidsequence of SEQ ID NO:
 120. 7. The method of claim 1, wherein the MEWclass I molecule is HLA-A*02.
 8. The method of claim 1, wherein thepopulation of transformed cells are administered in the form of apharmaceutical composition.
 9. The method of claim 8, wherein thepharmaceutical composition comprises a chemotherapeutic agent selectedfrom the group consisting of asparaginase, busulfan, carboplatin,cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine,hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, andvincristine.
 10. The method of claim 1, wherein the TCR comprises: aCDR1α chain comprising the amino acid sequence of SEQ ID NO: 1, a CDR2αchain comprising the amino acid sequence of SEQ ID NO: 2, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO:
 3. a CDR1β chaincomprising the amino acid sequences of SEQ ID NO: 7, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 8, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO: 9, and wherein each ofSEQ ID NOs: 2 and 8 comprises at most one conservative amino acidsubstitution.
 11. The method of claim 1, wherein the TCR comprises: aCDR1α chain comprising the amino acid sequence of SEQ ID NO: 13, a CDR2αchain comprising the amino acid sequence of SEQ ID NO: 14, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO: 15, a CDR1β chaincomprising the amino acid sequence of SEQ ID NO: 19, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 20, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO: 21, and wherein each ofSEQ ID NOs: 14 and 20 comprises at most one conservative amino acidsubstitution.
 12. The method of claim 1, wherein the TCR comprises: aCDR1α chain comprising the amino acid sequence of SEQ ID NO: 25, a CDR2αchain comprising the amino acid sequence of SEQ ID NO: 26, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO: 27, a CDR1β chaincomprising the amino acid sequence of SEQ ID NO: 31, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 32, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO: 33, and wherein each ofSEQ ID NOs: 26 and 32 comprises at most one conservative amino acidsubstitution.
 13. The method of claim 1, wherein the TCR comprises: aCDR1α chain comprising the amino acid sequence of SEQ ID NO: 37, a CDR2αchain comprising the amino acid sequence of SEQ ID NO: 38, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO: 39, a CDR1β chaincomprising the amino acid sequence of SEQ ID NO: 43, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 44, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO: 45, and wherein each ofSEQ ID NOs: 38 and 44 comprises at most one conservative amino acidsubstitution.
 14. The method of claim 1, wherein the TCR comprises: aCDR1α chain comprising the amino acid sequence of SEQ ID NO: 49, a CDR2αchain comprising the amino acid sequence of SEQ ID NO: 50, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO: 51, a CDR1β chaincomprising the amino acid sequence of SEQ ID NO: 55, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 56, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO: 57, and wherein each ofSEQ ID NOs: 50 and 56 comprises at most one conservative amino acidsubstitution.
 15. The method of claim 1, wherein the TCR comprises: aCDR1α chain comprising the amino acid sequence of SEQ ID NO: 61, a CDR2αchain comprising the amino acid sequence of SEQ ID NO: 62, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO: 63, a CDR1β chaincomprising the amino acid sequence of SEQ ID NO: 67, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 68, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO: 69, and wherein each ofSEQ ID NOs: 62 and 68 comprises at most one conservative amino acidsubstitution.
 16. The method of claim 1, wherein the TCR comprises: aCDR1α chain comprising the amino acid sequence of SEQ ID NO: 73, a CDR2αchain comprising the amino acid sequence of SEQ ID NO: 74, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO: 75, a CDR1β chaincomprising the amino acid sequence of SEQ ID NO: 79, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 80, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO: 81, and wherein each ofSEQ ID NOs: 74 and 80 comprises at most one conservative amino acidsubstitution.
 17. The method of claim 1, wherein the TCR comprises: aCDR1α chain comprising the amino acid sequence of SEQ ID NO: 85, a CDR2αchain comprising the amino acid sequence of SEQ ID NO: 86, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO: 87, a CDR1β chaincomprising the amino acid sequence of SEQ ID NO: 91, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 92, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO: 93, and wherein each ofSEQ ID NOs: 86 and 92 comprises at most one conservative amino acidsubstitution.
 18. The method of claim 1, wherein the TCR comprises: aCDR1α chain comprising the amino acid sequence of SEQ ID NO: 97, a CDR2αchain comprising the amino acid sequence of SEQ ID NO: 98, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO: 99, a CDR1β chaincomprising the amino acid sequences of SEQ ID NO: 103, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 104, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO: 105, and wherein eachof SEQ ID NOs: 98 and 104 comprises at most one conservative amino acidsubstitution.
 19. The method of claim 1, wherein the TCR comprises: aCDR1α chain comprising the amino acid sequence of SEQ ID NO: 109, aCDR2α chain comprising the amino acid sequence of SEQ ID NO: 110, aCDR3α chain comprising the amino acid sequence of SEQ ID NO: 111, aCDR1β chain comprising the amino acid sequence of SEQ ID NO: 115, aCDR2β chain comprising the amino acid sequence of SEQ ID NO: 116, and aCDR3β chain comprising the amino acid sequence of SEQ ID NO: 117, andwherein each of SEQ ID NOs: 110 and 116 comprises at most oneconservative amino acid substitution.
 20. The method of claim 1, whereinthe TCR comprises: a CDR1α chain comprising the amino acid sequence ofSEQ ID NO: 1, a CDR2α chain comprising the amino acid sequence of SEQ IDNO: 2, a CDR3α chain consisting of the amino acid sequence of SEQ ID NO:3, a CDR1β chain comprising the amino acid sequence of SEQ ID NO: 7, aCDR2β chain comprising the amino acid sequence of SEQ ID NO: 8, and aCDR3β chain consisting of the amino acid sequence of SEQ ID NO: 9.